Method of improving the properties of a flour dough, a flour dough improving composition and improved food products

ABSTRACT

A method of improving the rheological and/or machineability properties of a flour dough and/or the quality of the product made from the dough, comprising adding to the dough a combination comprising a Hox and an emulsifying agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of the filing date ofU.S. Provisional Patent Application No. 60/398,020, filed Jul. 24, 2002.This application is also a continuation-in-part of U.S. patentapplication Ser. No. 09/932,923, filed Aug. 21, 2001, now U.S. Pat. No.6,726,942, which is a continuation of application Ser. No. 08/676,186filed Sep. 12, 1996, now U.S. Pat. No. 6,358,543, which was acontinuation-in-part of application Ser. No. 08/483,870, filed Jun. 7,1995, abandoned. The Ser. No. 08/483,870 application was a U.S. nationalphase of PCT/DK96/00239, filed Jun. 4, 1996. This application isadditionally a continuation-in-part of application Ser. No. 10/040,394,filed Jan. 9, 2002, now U.S. Pat. No. 6,852,346, which was a divisionalof application Ser. No. 09/402,664, filed Oct. 22, 1999, now U.S. Pat.No. 6,406,723. U.S. Pat. No. 6,406,723 was the U.S. national phase ofPCT/DK98/00136, filed Apr. 3, 1998, which claimed priority fromDK0400/97, filed Apr. 9, 1997. This application is also acontinuation-in-part of U.S. application Ser. No. 10/150,429, filed May17, 2002, now U.S. Pat. No. 6,967,035, which claims priority from UKApplication 0112226.6, filed May 18, 2001, and U.S. Application No.60/347,007, filed Jan. 9, 2002. We claim the benefit of priority datesof all the above applications. This application also claims the benefitof priority of the filing date of Great Britain Application 0211975.8,filed May 24, 2002. The contents of all of the above applications areincorporated herein by reference to the extent they are consistent withthis application and inventions described herein.

FIELD OF INVENTION

The invention pertains to the provision of flour doughs having improvedrheological properties and farinaceous food products having improvedquality characteristics and it provides a maltose oxidizingoxidoreductase-containing compo-sition capable of conferring suchimproved properties on doughs and finished food products made herefromwhen it is added as a component to the doughs, and a method of preparingimproved doughs and farinaceous food products.

More in particular, the present invention relates to the field of foodmanufacturing, in particular to the preparation of improved bakeryproducts and other farinaceous food products. Specifically, theinvention concerns the use of a new combination for improving thestability and/or machineability of dough and/or improving the quality ofbaked and dried products made from such doughs.

In a preferred aspect, the present invention relates to:

-   -   a method of improving the rheological and/or machineability        properties of a flour dough and/or the quality of the product        made from the dough, comprising adding to the dough a        combination comprising a Hox and an emulsifying agent.

Teachings relating to this preferred aspect now follow.

TECHNICAL BACKGROUND AND PRIOR ART

The invention relates in particular to a method of providing flourdoughs having improved rheological properties and to finished baked ordried products made from such doughs, which have improved textural,eating quality and dimensional characteristics.

In this connection, the “strength” or “weakness” of doughs is animportant aspect of making farinaceous finished products from doughs,including baking. The “strength” or “weakness” of a dough is primarilydetermined by its content of protein and in particular the content andthe quality of the gluten protein is an important factor in thatrespect. Flours with a low protein content are generally characterizedas “weak”. Thus, the cohesive, extensible, rubbery mass which is formedby mixing water and weak flour will usually be highly extensible whensubjected to stress, but it will not return to its original dimensionswhen the stress is removed.

Flours with a high protein content are generally characterized as“strong” flours and the mass formed by mixing such a flour and waterwill be less extensible than the mass formed from a weak flour, andstress which is applied during mixing will be restored without breakdownto a greater extent than is the case with a dough mass formed from aweak flour.

Strong flour is generally preferred in most baking contexts because ofthe superior rheological and handling properties of the dough and thesuperior form and texture qualities of the finished baked or driedproducts made from the strong flour dough.

Dough quality may be largely dependent on the type or types of flourpresent in the dough and/or the age of the flour or flours.

Doughs made from strong flours are generally more stable. Stability of adough is one of the most important characteristics of flour doughs.According to American Association of Cereal Chemists (AACC) Method36-01A the term “stability” can be defined as “the range of dough timeover which a positive response is obtained and that property of arounded dough by which it resists flattening under its own weight over acourse of time”. According to the same method, the term “response” isdefined as “the reaction of dough to a known and specific stimulus,substance or set of conditions, usually determined by baking it incomparison with a control”

Within the bakery and milling industries it is known to use dough“conditioners” to strengthen the dough to increase its stability andstrength. Such dough conditioners are normally non-specific oxidizingagents such as eg iodates, peroxides, ascorbic acid, K-bromate orazodi-carbonamide and they are added to dough with the aims of improvingthe baking performance of flour to achieve a dough with improvedstretchability and thus having a desirable strength and stability. Themechanism behind this effect of oxidizing agents is that the flourproteins, in particular gluten contains thiol groups which, when theybecome oxidized, form disulphide bonds whereby the protein forms a morestable matrix resulting in a better dough quality and improvements ofthe volume and crumb structure of the baked products.

In addition to the above usefulness of ascorbic acid/ascorbate as adough conditioner due to its oxidizing capacity, these compounds mayalso act as substrate for an oxidoreductase, ascorbate oxidase which isdisclosed in EP 0 682 116 A1. In the presence of its substrate, thisenzyme converts ascorbic acid/ascorbate to dehydroascorbic acid andH₂O₂. This prior art does not suggest that ascorbic acid oxidase in thepresence of ascorbic acid/ascorbate might have a dough conditioningeffect, but assumingly this is the case.

However, the use of several of the currently available oxidizing agentsis either objected to by consumers or is not permitted by regulatorybodies and accordingly, it has been attempted to find alternatives tothese conventional flour and dough additives and the prior art has i.a.suggested the use of glucose oxidase for this purpose. In addition, theprior art has inter alia (i.a.) suggested the use of oxidoreductasessuch as carbohydrate oxidase, glycerol oxidase and hexose oxidase forthis purpose.

Thus, U.S. Pat. No. 2,783,150 discloses the addition of glucose oxidaseto flour to improve dough strength and texture and appearance of bakedbread.

CA 2,012,723 discloses bread improving compositions comprisingcellulolytic enzymes such as xylanases and glucose oxidase, the latterenzyme being added to reduce certain disadvantageous effects of thecellulolytic enzymes (reduced dough strength and stickiness) and it isdisclosed that addition of glucose to the dough is required to obtain asufficient glucose oxidase activity.

JP-A-92-084848 suggests the use of a bread improving compositioncomprising glucose oxidase and lipase.

EP-B1-321 811 discloses the use of an enzyme composition comprisingsulfhydryl oxidase and glucose oxidase to improve the rheologicalcharacteristics of doughs. It is mentioned in this prior art documentthat the use of glucose oxidase alone has not been successful.

In EP-B1-338 452 is disclosed an enzyme composition for improving doughstability, comprising a mixture of cellulase/hemicellulase, glucoseoxidase and optionally sulfhydryl oxidase.

However, the use of glucose oxidase as a dough improving additive hasthe limitation that this enzyme requires the presence of sufficientamounts of glucose as substrate in order to be effective in a doughsystem and generally, the glucose content in cereal flours is low.Therefore, the absence of glucose in doughs or the low content hereof indoughs will be a limiting factor for the effectiveness of glucoseoxidase as a dough improving agent.

In contrast hereto, the content of maltose in cereal flours is generallysignificantly higher than that of glucose and accordingly, a freshlyprepared dough will normally contain more maltose than glucose. Thus, inan experiment where the content of sugars in supernatants fromsuspensions of wheat flour and a dough prepared from the flour andfurther comprising water, yeast, salt and sucrose (as described in thefollowing example 2.3) were analyzed, the following values (% by weightcalculated on flour) were found:

Flour Dough Sucrose 0.3 <0.01 Galactose 0.001 0.01 Glucose 0.25 0.72Maltose 2.6 1.4 Fructose 0.06 0.67 Lactose <0.01 <0.01

In addition, the content of maltose remains at a relatively high levelin a dough which is leavened by yeast, since the yeast primarilyutilizes glucose, or it may even increase in the dough e.g. duringproofing due to the activity of starch degrading enzymes such as e.g.β-amylase, which is inherently present in the flour or is added to thedough.

Whereas the prior art has recognized the useful improving effects ofglucose oxidase on the rheological characteristics of bread doughs andon the quality of the corresponding baked products, it has also beenrealized that the use of this enzyme has several drawbacks. Thus, it maybe required to add sucrose or glucose as substrate to the dough toobtain a sufficient effect and glucose oxidase does not constantlyprovide a desired dough or bread improving effect when used alonewithout the addition of other enzymes.

However, it has now been found that the addition of an oxidoreductase,which is capable of oxidizing maltose, including hexose oxidase as asole dough conditioning agent, i.e. without concomitant addition ofsubstrate for the added enzyme, or of other enzymes, to a farinaceousdough results in an increased resistance hereof to breaking when thedough is stretched, i.e. this enzyme confers in itself to the dough anincreased strength whereby the dough becomes less prone to mechanicaldeformation. It is contemplated that this effect of addition of hexoseoxidase to a dough is the result of the formation of cross-links betweenthiol groups in sulphur-containing amino acids in wheat gluten whichoccurs when the H₂O₂ generated by the enzyme in the dough reacts withthe thiol groups which are hereby oxidized.

Hexose oxidase (D-hexose: O₂-oxidoreductase, EC 1.1.3.5) is an enzymewhich in the presence of oxygen is capable of oxidizing D-glucose andseveral other reducing sugars including maltose, glucose, lactose,galactose, xylose, arabinose and cellobiose to their correspondinglactones with subsequent hydrolysis to the respective aldobionic acids.Accordingly, hexose oxidases differ from glucose oxidase which can onlyconvert D-glucose, in that hexose oxidases can utilize a broader rangeof sugar substrates. The oxidation catalyzed by the enzyme can beillustrated as follows:

D-Glucose+O₂->δ-D-gluconolactone+H₂O₂, or

D-Galactose+O₂->γ-D-galactogalactone+H₂O₂

Hexose oxidase (in the following also referred to as HOX) has beenisolated from several red algal species such as Iridophycus flaccidum(Bean and Hassid, 1956, J. Biol. Chem., 218:425-436) and Chondruscrispus (Ikawa 1982, Methods Enzymol., 89:145-149). Additionally, thealgal species Euthora cristata (Sullivan et al. 1973, Biochemica etBiophysica Acta, 309:11-22) has been shown to produce HOX.

Other potential sources of hexose oxidase according to the inventioninclude microbial species or land-growing plant species. Thus, as anexample of such a plant source, Bean et al., Journal of BiologicalChemistry (1961) 236: 1235-1240, have disclosed an oxidoreductase fromcitrus fruits which is capable of oxidizing a broad range of sugarsincluding D-glucose, D-galactose, cellobiose, lactose, maltose,D-2-deoxyglucose, D-mannose, D-glucosamine and D-xylose. Another exampleof an enzyme having hexose oxidase activity is the enzyme system ofMalleomyces mallei disclosed by Dowling et al., Journal of Bacteriology(1956) 72:555-560.

It has been reported that hexose oxidase isolated from the above naturalsources may be of potential use in the manufacturing of certain foodproducts. Thus, hexose oxidase isolated from Iridophycus flaccidum hasbeen shown to be capable of converting lactose in milk with theproduction of the corresponding aldobionic acid and has been shown to beof potential interest as an acidifying agent in milk, e.g. to replaceacidifying microbial cultures for that purpose (Rand, 1972, Journal ofFood Science, 37:698-701). In that respect, hexose oxidase has beenmentioned as a more interesting enzyme than glucose oxidase, since thislatter enzyme can only be enzymatically effective in milk or other foodproducts not containing glucose or having a low content of glucose, ifglucose or the lactose-degrading enzyme lactase which convert thelactose to glucose and galactose, is also added.

The capability of oxidoreductases including that of hexose oxidase togenerate hydrogen peroxide has also been utilized to improve the storagestability of certain food products including cheese, butter and fruitjuice as it is disclosed in JP-B-73/016612. It has also been suggestedthat oxidoreductases may be potentially useful as antioxidants in foodproducts.

However, the present invention has demonstrated that hexose oxidase ishighly useful as a dough conditioning agent in the manufacturing offlour dough products including not only bread products but also otherproducts made from flour doughs such as noodles and alimentary pasteproducts.

WO 94/04035 discloses a method of improving properties of a dough (withand without fat) and/or baked product made from dough by adding a lipaseof microbial origin to the dough. The use of the microbial lipaseresulted in an increased volume and improved softness of the bakedproduct. Furthermore an antistaling effect was found.

EP 1 108 360 A1 discloses a method of preparing a flour dough. Themethod comprises adding to the dough components an enzyme that underdough conditions is capable of hydrolysing a nonpolar lipid, aglycolipid and a phospholipid, or a composition containing said enzymeand mixing the dough components to obtain the dough.

WO 02/03805 discloses that the addition to dough of a combination of twolipases with different substrate specificities. The combination producesa synergistic effect on the dough or on a baked product made from thedough. Optionally, an additional enzyme may be used together with thelipase.

SUMMARY OF THE INVENTION

Accordingly, the invention relates in a first aspect to a method ofimproving the rheological properties of a flour dough and the quality ofthe finished product made from the dough, comprising adding to the doughingredients, dough additives or the dough an effective amount of anoxidoreductase which at least is capable of oxidizing maltose, such ase.g. a hexose oxidase.

In a further aspect, there is also provided a dough bakery productimproving composition comprising an oxidoreductase which at least iscapable of oxidizing maltose, and at least one further dough ingredientor dough additive.

In still further aspects, the invention pertains to a method ofpreparing a bakery product, comprising preparing a flour dough includingadding an effective amount of an oxidoreductase which at least iscapable of oxidizing maltose and baking the dough, and a method ofpreparing a dough-based food product comprising adding to the dough aneffective amount of a maltose oxidizing oxidoreductase.

In addition, we have surprisingly found that a combination of a Hox andan emulsifying agent results in particularly advantageous properties indough and dough products and/or in baked products therefrom. Inparticular the stability (e.g. shock stability) and/or rheological (e.g.decrease in stickiness) and/or machineability properties and/or theresultant volume of either the dough and/or baked products (e.g. bakedproducts with better crumb structure and/or homogeneity) is/areimproved. Furthermore, the combination of the Hox and emulsifying agentresults in an improvement in bread quality, in particular in respect ofspecific volume and/or crumb homogeneity, which is not a simple additiveeffect, but may reflect a synergistic effect of these types of enzymes.

The invention further relates to the use of a Hox and an emulsifyingagent to improve the rheological and/or machineability properties ofdough.

The invention further relates to the use of a Hox and an emulsifyingagent to improve the volume of a baked product made from a dough.

DETAILED DISCLOSURE OF THE INVENTION

In one aspect, the present method contemplates a method of improving therheological properties of flour doughs. The method comprises, as it ismentioned above, the addition of an effective amount of a maltoseoxidizing oxidoreductase either to a component of the dough recipe or tothe dough resulting from mixing all of the components for the dough. Inthe present context, “an effective amount” is used to indicate that theamount is sufficient to confer to the dough and/or the finished productimproved characteristics as defined herein.

In another aspect the invention provides a method of improving therheological and/or machineability properties of a flour dough and/or thequality (e.g. volume) of the product made from the dough, comprisingadding to the dough a combination comprising a Hox and an emulsifyingagent.

Factors which influence the rheological properties and/or themachineability include stickiness and extensibility.

In another aspect the invention provides a method of improving thetheological and/or machineability properties of a flour dough and/or thequality (e.g. volume) of the product made from the dough, comprisingadding to the dough a combination comprising a Hox and an emulsifyingagent wherein the flour dough comprises at least one further doughadditive or ingredient.

In another aspect the invention provides a method of improving therheological and/or machineability properties of a flour dough and/or thequality (e.g. volume) of the product made from the dough, comprisingadding to the dough a combination comprising a Hox and an emulsifyingagent wherein the product is selected from the group consisting of abread product, a noodle product, a cake product, a pasta product and analimentary paste product.

In another aspect the invention provides a method of improving therheological and/or machineability properties of a flour dough and/or thequality (e.g. volume) of the product made from the dough, comprisingadding to the dough a combination comprising a Hox and an emulsifyingagent wherein at least one further enzyme is added to the doughingredients, dough additives or the dough.

In another aspect the invention provides a dough improving compositioncomprising a Hox and an emulsifying agent.

In another aspect the invention provides a dough improving compositioncomprising a Hox and an emulsifying agent wherein the flour doughcomprises at least one further dough additive or ingredient.

In another aspect the invention provides use of a dough improvingcomposition comprising a Hox and an emulsifying agent in the manufactureof a product made from dough wherein the product is selected from thegroup consisting of a bread product, a noodle product, a cake product, apasta product and an alimentary paste product.

In another aspect the invention provides a dough improving compositioncomprising a Hox and an emulsifying agent wherein at least one furtherenzyme is added to the dough ingredients, dough additives or the dough.

In another aspect the invention provides use of a dough improvingcomposition comprising a Hox and an emulsifying agent wherein saidcomposition improves the rheological and/or machineability properties offlour dough.

In another aspect the invention provides use of a dough improvingcomposition comprising a Hox and an emulsifying agent wherein saidcomposition improves the volume of a baked product made from a flourdough.

In another aspect the invention provides a dough for addition to asponge wherein said dough comprises a Hox and an emulsifying agent.

In another aspect the invention provides a dough for addition to asponge wherein said dough comprises a Hox and an emulsifying agent andwherein the dough comprises at least one further dough additive oringredient.

Hexose Oxidase

In one useful embodiment of the method according to the invention, theoxidoreductase is a hexose oxidase.

The term “Hox” as used herein refers to Hexose oxidase(D-hexose:O₂-oxidoreductase, EC 1.1.3.5). Below discloses some of thesources of Hox. WO 96/40935 discloses a method of producing Hox byrecombinant DNA technology. U.S. Pat. No. 6,251,626 discloses hexoseoxidase sequences.

The Hox may be isolated and/or purified from natural sources or it maybe prepared by use of recombinant DNA techniques.

The Hox may be a variant or derivative of a natural Hox.

The Hox, or the variant or derivative of a natural Hox, is capable ofoxidising maltose in the dough.

Preferably the Hox is added in a substantially pure and/or substantiallyisolated form.

Hexose oxidase can, as it is described in details herein, be isolatedfrom marine algal species naturally producing that enzyme. Such speciesare found in the family Gigartinaceae which belong to the orderGigartinales. Examples of hexose oxidase producing algal speciesbelonging to Gigartinaceae are Chondrus crispus and Iridophycusflaccidum. Also algal species of the order Cryptomeniales including thespecies Euthora cristata are potential sources of hexose oxidase.

When using such natural sources for hexose oxidase, the enzyme istypically isolated from the algal starting material by extraction usingan aqueous extraction medium. As starting material may be used algae intheir fresh state as harvested from the marine area where they grow, orthe algal material can be used for extraction of hexose oxidase afterdrying the fronds e.g. by air-drying at ambient temperatures or by anyappropriate industrial drying method such as drying in circulated heatedair or by freeze-drying. In order to facilitate the subsequentextraction step, the fresh or dried starting material may advantageouslybe comminuted e.g. by grinding or blending.

As the aqueous extraction medium, buffer solutions e.g. having a pH inthe range of 5-8, such as 0.1 M sodium phosphate buffer, 20 mMtriethanolamine buffer or 20 mM Tris-HCl buffer are suitable. The hexoseoxidase is typically extracted from the algal material by suspending thestarting material in the buffer and keeping the suspension at atemperature in the range of 0-20° C. such as at about 5° C. for 1 to 5days, preferably under agitation.

The suspended algal material is then separated from the aqueous mediumby an appropriate separation method such as filtration, sieving orcentrifugation and the hexose oxidase is subsequently recovered from thefiltrate or supernatant. Optionally, the separated algal material issubjected to one or more further extraction steps.

Since several marine algae contain coloured pigments such asphycocyanins, it may be required to subject the filtrate or supernatantto a further purification step whereby these pigments are removed. As anexample, the pigments may be removed by treating the filtrate orsupernatant with an organic solvent in which the pigments are solubleand subsequently separating the solvent containing the dissolvedpigments from the aqueous medium. Alternatively, pigments may be removedby subjecting the filtrate or supernatant to a hydrophobic interactionchromatography step.

The recovery of hexose oxidase from the aqueous extraction medium iscarried out by any suitable conventional methods allowing isolation ofproteins from aqueous media. Such methods, examples of which will bedescribed in details in the following, include conventional methods forisolation of proteins such as ion exchange chromatography, optionallyfollowed by a concentration step such as ultrafiltration. It is alsopossible to recover the enzyme by adding substances such as e.g.(NH₄)₂SO₄ or polyethylene glycol (PEG) which causes the protein toprecipitate, followed by separating the precipitate and optionallysubjecting it to conditions allowing the protein to dissolve.

For certain applications of hexose oxidase it is desirable to providethe enzyme in a substantially pure form e.g. as a preparationessentially without other proteins or non-protein contaminants andaccordingly, the relatively crude enzyme preparation resulting from theabove extraction and isolation steps may be subjected to furtherpurification steps such as further chromatography steps, gel filtrationor chromatofocusing as it will also be described by way of example inthe following.

Further Dough Additives or Ingredients (Components)

In a preferred embodiment of the method according to the invention, aflour dough is prepared by mixing flour with water, a leavening agentsuch as yeast or a conventional chemical leavening agent, and aneffective amount of hexose oxidase under dough forming conditions. Itis, however, within the scope of the invention that further componentscan be added to the dough mixture.

Typically, such further dough components include conventionally useddough components such as salt (such as sodium chloride, calcium acetate,sodium sulfate or calcium sulfate), sweetening agents such as sugars,syrups or artificial sweetening agents, lipid substances includingshortening, margarine, butter or an animal or vegetable oil, glyceroland one or more dough additives such as emulsifying agents, starchdegrading enzymes, cellulose or hemicellulose degrading enzymes,proteases, lipases, non-specific oxidizing agents such as thosementioned above, flavouring agents, lactic acid bacterial cultures,vitamins, minerals, hydrocolloids such as alginates, carrageenans,pectins, vegetable gums including e.g. guar gum and locust bean gum, anddietary fiber substances.

The dough may also comprise other conventional dough ingredients, e.g.:proteins, such as milk powder, gluten, and soy; eggs (either whole eggs,egg yolks or egg white); an oxidant such as ascorbic acid, potassiumbromate, potassium iodate, azodicarbonamide (ADA) or ammoniumpersulfate; an amino acid such as L-cysteine; a sugar.

The dough may comprise fat such as granulated fat or shortening.

The further dough additive or ingredient can be added together with anydough ingredient including the flour, water or optional otheringredients or additives, or the dough improving composition. Thefurther dough additive or ingredient can be added before the flour,water, optional other ingredients and additives or the dough improvingcomposition. The further dough additive or ingredient can be added afterthe flour, water, optional other ingredients and additives or the doughimproving composition.

The further dough additive or ingredient may conveniently be a liquidpreparation. However, the further dough additive or ingredient may beconveniently in the form of a dry composition.

Preferably the further dough additive or ingredient is selected from thegroup consisting of a vegetable oil, a vegetable fat, an animal fat,shortening, glycerol, margarine, butter, butterfat and milk fat.

Preferably the further dough additive or ingredient is at least 1% theweight of the flour component of dough. More preferably, the furtherdough additive or ingredient is at least 2%, preferably at least 3%,preferably at least 4%, preferably at least 5%, preferably at least 6%.

If the additive is a fat, then typically the fat may be present in anamount of from 1 to 5%, typically 1 to 3%, more typically about 2%.

Further Enzymes

In one advantageous embodiment of the above method at least one furtherenzyme is added to the dough. Suitable examples hereof include acellulase, a hemicellulase, a xylanase, a starch degrading enzyme, aglucose oxidase, a lipase, a lipoxygenase, an oxidoreductase and aprotease.

Among starch degrading enzymes, amylases are particularly useful asdough improving additives. Other useful starch degrading enzymes whichmay be added to a dough composition include glucoamylases andpullulanases.

The term “xylanase” as used herein refers to xylanases (EC 3.2.1.32)which hydrolyse xylosidic linkages.

The further enzyme can be added together with any dough ingredientincluding the flour, water or optional other ingredients or additives,or the dough improving composition. The further enzyme can be addedbefore the flour, water, and optionally other ingredients and additivesor the dough improving composition. The further enzyme can be addedafter the flour, water, and optionally other ingredients and additivesor the dough improving composition.

The further enzyme may conveniently be a liquid preparation. However,the composition may be conveniently in the form of a dry composition.

In some aspects of the present invention it may be found that someenzymes of the dough improving composition of the invention are capableof interacting with each other under the dough conditions to an extentwhere the effect on improvement of the rheological and/or machineabilityproperties of a flour dough and/or the quality of the product made fromdough by the enzymes is not only additive, but the effect issynergistic.

In relation to improvement of the product made from dough (finishedproduct), it may be found that the combination results in a substantialsynergistic effect in respect to crumb homogeneity as defined herein.Also, with respect to the specific volume of baked product a synergisticeffect may be found.

Emulsifying Agent

The dough may further comprise a further emulsifier such as mono- ordiglycerides, sugar esters of fatty acids, polyglycerol esters of fattyacids, lactic acid esters of monoglycerides, acetic acid esters ofmonoglycerides, polyoxethylene stearates, or lysolecithin. Among starchdegrading enzymes, amylases are particularly useful as dough improvingadditives. α-amylase breaks down starch into dextrins which are furtherbroken down by β-amylase into maltose. Other useful starch degradingenzymes which may be added to a dough composition include glucoamylasesand pullulanases. In the present context, further interesting enzymesare xylanases and other oxidoreductases such as glucose oxidase,pyranose oxidase and sulfhydryl oxidase.

Conventional emulsifiers used in making flour dough products include asexamples monoglycerides, diacetyl tartaric acid esters of mono- anddiglycerides of fatty acids, and lecithins e.g. obtained from soya.

The emulsifying agent may be an emulsifier per se or an agent thatgenerates an emulsifier in situ.

Examples of emulsifying agents that can generate an emulsifier in situinclude enzymes.

Preferably the emulsifying agent is a lipase.

Lipase

The term “lipase” as used herein refers to enzymes which are capable ofhydrolysing carboxylic ester bonds to release carboxylate (EC 3.1.1).Examples of lipases include but are not limited to triacylglycerollipase (EC 3.1.1.3), galactolipase (EC 3.1.1.26), phospholipase (EC3.1.1.32).

The lipase may be isolated and/or purified from natural sources or itmay be prepared by use of recombinant DNA techniques.

Preferably the lipase is selected from the group comprisingtriacylglycerol lipase, a galactolipase, phospholipase.

More preferably the lipase(s) may be one or more of: triacylglycerollipase (EC 3.1.1.3), phospholipase A2 (EC 3.1.1.4), galactolipase (EC3.1.1.26), phospholipase A1 (EC 3.1.1.32), lipoprotein lipase A2 (EC3.1.1.34).

The lipase may be a variant or derivative of a natural lipase.

For some aspects, preferably the lipase is a phospholipase (including avariant phospholipase).

Preferably the lipase is added in a substantially pure and/orsubstantially isolated form.

Lipases that are useful in the present invention can be derived from abacterial species, a fungal species, a yeast species, an animal cell anda plant cell. Whereas the enzyme may be provided by cultivating culturesof such source organisms naturally producing lipase, it may be moreconvenient and cost-effective to produce it by means of geneticallymodified cells such as it is described WO 9800136. The term “derived”may imply that a gene coding for the lipase is isolated from a sourceorganism and inserted into a host cell capable of expressing the gene.

WO 02/03805 teaches some of the sources of lipases. The lipases that aretaught therein are incorporated herein by reference.

For some aspects of the present invention the lipase may be Lipopan F(supplied by Novozymes) or a variant thereof.

Dough Preparation

The dough is prepared by admixing flour, water, the oxidoreductaseaccording to the invention or the dough improving composition andoptionally other possible ingredients and additives. The oxidoreductaseor dough improving composition can be added together with any doughingredient including the flour, the water or dough ingredient mixture orwith any additive or additive mixture. The dough improving compositioncan be added before the flour or water or optional other ingredients andadditives. The dough improving composition can be added after the flouror water, or optional other ingredients and additives. The dough can beprepared by any conventional dough preparation method common in thebaking industry or in any other industry making flour dough basedproducts.

The dough of the invention generally comprises wheat meal or wheat flourand/or other types of meal, flour or starch such as corn flour, cornstarch, maize flour, rice flour, rye meal, rye flour, oat flour, oatmeal, soy flour, sorghum meal, sorghum flour, potato meal, potato flouror potato starch.

A preferred flour is wheat flour, but doughs comprising flour derivedfrom other cereal species such as from rice, maize, corn, oat, barley,rye, durra, soy, sorghum and potato are also contemplated.

Preferably the flour dough comprises a hard flour.

The term “hard flour” as used herein refers to flour which has a higherprotein content such as gluten than other flours and is suitable for theproduction of, for example, bread. The term “hard flour” as used hereinis synonymous with the term “strong flour”.

Preferably the flour dough comprises a hard wheat flour.

The invention also provides a pre-mix comprising flour together with thecombination as described herein. The pre-mix may contain otherdough-improving and/or bread-improving additives, e.g. any of theadditives, including enzymes, mentioned herein.

The dough of the invention may be fresh, frozen, or part-baked.

The dough of the invention can be a leavened dough or a dough to besubjected to leavening. The dough may be leavened in various ways, suchas by adding chemical leavening agents, e.g., sodium bicarbonate or byadding a leaven (fermenting dough), but it is preferred to leaven thedough by adding a suitable yeast culture, such as a culture ofSaccharomyces cerevisiae (baker's yeast), e.g. a commercially availablestrain of S. cerevisiae.

The oxidoreductase or the dough improving composition can be added as aliquid preparation or in the form of a dry powder composition eithercomprising the enzyme as the sole active component or in admixture withone or more other dough ingredients or additive.

The amount of the enzyme component added normally is an amount whichresults in the presence in the finished dough of 1 to 10,000 units perkg of flour, preferably 5 to 5000 units such as 10 to 1000 units. Inuseful embodiments, the amount is in the range of 20 to 500 units per kgof flour. In the present context 1 oxidoreductase unit corresponds tothe amount of enzyme which under specified conditions results in theconversion of 1 μmole glucose per minute. The activity is stated asunits per g of enzyme preparation.

Rheological Properties

The phrase “rheological properties” as used herein relates to thephysical and chemical phenomena described herein which in combinationwill determine the performance of flour doughs and thereby also thequality of the resulting products.

The phrase “machineability of a flour dough” as used herein refers tothe improved manipulation by machinery of the dough. The dough is lesssticky compared to the dough without the addition of the combination.

In a further embodiment, the invention relates to improvement of therheological characteristics of the dough including that the gluten indexin the dough is increased by at least 5%, relative to a dough withoutaddition of a combination, the gluten index is determined by means of aGlutomatic 2200 apparatus.

The phrase “rheological properties” as used herein refers to the effectsof dough conditioners on dough strength and stability as the mostimportant characteristics of flour doughs. According to AmericanAssociation of Cereal Chemists (AACC) Method 36-01A the term “stability”can be defined as “the range of dough time over which a positiveresponse is obtained and that property of a rounded dough by which itresists flattening under its own weight over a course of time”.According to the same method, the term “response” is defined as “thereaction of dough to a known and specific stimulus, substance or set ofconditions, usually determined by baking it in comparison with acontrol”.

As it is mentioned herein, it is generally desirable to improve thebaking performance of flour to achieve a dough with improvedstretchability and thus having a desirable strength and stability byadding oxidising agents which cause the formation of protein disulphidebonds whereby the protein forms a more stable matrix resulting in abetter dough quality and improvements of the volume and crumb structureof baked products.

The effect of the oxidoreductase or the dough improving composition onthe rheological properties of the dough can be measured by standardmethods according to the International Association of Cereal Chemistry(ICC) and the American Association of Cereal Chemistry (AACC) includingthe amylograph method (ICC 126), the farinograph method (AACC 54-21) andthe extensigraph method (AACC 54-10). The extensigraph method measurese.g. the doughs ability to retain gas evolved by yeast and the abilityto withstand proofing. In effect, the extensigraph method measures therelative strength of a dough. A strong dough exhibits a higher and, insome cases, a longer extensigraph curve than does a weak dough. AACCmethod 54-10 defines the extensigraph in the following manner: “theextensigraph records a load-extension curve for a test piece of doughuntil it breaks. Characteristics of load-extension curves orextensigrams are used to assess general quality of flour and itsresponses to improving agents”.

In a preferred embodiment of the invention, the resistance to extensionof the dough in terms of the ratio between the resistance to extension(height of curve, B) and the extensibility (length of curve, C), i.e.the B/C ratio as measured by the AACC method 54-10 is increased by atleast 10% relative to that of an otherwise similar dough not containingoxidoreductase. In more preferred embodiments, the resistance toextension is increased by at least 20%, such as at least 50% and inparticular by at least 100%.

The method according to the invention can be used for any type of flourdough with the aims of improving the rheologi-cal properties hereof andthe quality of the finished prod-ucts made from the particular type ofdough. Thus, the method is highly suitable for the making ofconventional types of yeast leavened bread products including wheatflour based bread products such as loaves and rolls. However, it iscontemplated that the method also can improve the properties of doughsin which leavening is caused by the addition of chemical leaveningagents, including sweet bakery products such as cake products includingas examples pound cakes and muffins, or scones.

Noodles

In one interesting aspect, the invention is used to improve therheological properties of doughs intended for noodle products including“white noodles” and “chinese noodles” and to improve the texturalqualities of the finished noodle products. A typical basic recipe forthe manufacturing of noodles comprises the following ingredients: wheatflour 100 parts, salt 0.5 parts and water 33 parts. Furthermore,glycerol is often added to the noodle dough. The noodles are typicallyprepared by mixing the ingredients in an appropriate mixing apparatusfollowed by rolling out the noodle dough using an appropriate noodlemachine to form the noodle strings which are subsequently air dried.

The quality of the finished noodles is assessed inter alia (i.a.) bytheir colour, cooking quality and texture. The noodles should cook asquickly as possible, remain firm after cooking and should preferably notloose any solids to the cooking water. On serving the noodles shouldpreferably have a smooth and firm surface not showing stickiness andprovide a firm “bite” and a good mouthfeel. Furthermore, it is importantthat the noodles have a light colour.

Since the appropriateness of wheat flour for providing noodles havingthe desired textural and eating qualities may vary according to the yearand the growth area, it is usual to add noodle improvers to the dough inorder to compensate for sub-optimal quality of the flour. Typically,such improvers will comprise dietary fiber substances, vegetableproteins, emulsifiers and hydrocolloids such as e.g. alginates,carrageenans, pectins, vegetable gums including guar gum and locust beangum, and amylases, and glycerol.

It has been attempted to use glucose oxidase as a noodle improvingagent. However, as mentioned above, the content of glucose may be so lowin wheat flour that this enzyme will not be effective.

It is therefore an important aspect of the invention that theoxidoreductase according to the invention and the composition accordingto the invention is useful as a noodle improving agent, optionally incombination with glycerol and other components currently used to improvethe quality of noodles. Thus, it is contemplated that noodles preparedin accordance with the above method will have improved properties withrespect to colour, cooking and eating qualities including a firm,elastic and non-sticky texture and consistency.

Alimentary Paste Product

In a further useful embodiment the dough which is prepared by the methodaccording to the invention is a dough for preparing an alimentary pasteproduct. Such products which include as examples spaghetti and maccaroniare typically prepared from a dough comprising as the main ingredientssuch as flour, eggs or egg powder and/or water. After mixing of theingredient, the dough is formed to the desired type of paste product andair dried. It is contemplated that the addition of the combination to apaste dough, optionally in combination with its substrate, will have asignificant improving effect on the extensibility and stability hereofresulting in finished paste product having textural and eatingqualities.

In a further aspect of the invention there is provided a dough improvingcomposition comprising the oxidoreductase according to the invention andat least one further dough ingredient or dough additive.

Bread

In the invention the improvement of the rheological properties of thedough include that the resistance to extension of the dough in terms ofthe ratio between resistance to extension (height of curve, B) and theextensibility (length of curve, C), i.e. the B/C ratio, as measured bythe AACC method 54-10 is increased by at least 10% relative to that ofan otherwise similar dough that does not comprise the combination andwherein the improvement of the quality of the finished product made fromthe dough is that the average pore diameter of the crumb of the breadmade from the dough is reduced by at least 10%, relative to a breadwhich is made from a bread dough without addition of the combination.

In a further embodiment, the invention, implies that the improvement ofthe quality of the product made from the dough consists in that the porehomogeneity of the crumb of the bread made from the dough is increasedby at least 5%, relative to a bread which is made from a bread doughwithout addition of the combination. The pore homogeneity of bread isconveniently measured by means of an image analyser composed of astandard CCD-video camera, a video digitiser and a personal computerwith WinGrain software. Using such an analyzer, the results of porediameter in mm and pore homogeneity can be calculated as an average ofmeasurements from 10 slices of bread. The pore homogeneity is expressedin % of pores that are larger than 0.5 times the average of porediameter and smaller than 2 times the average diameter.

Preferably, the dough is a yeast leavened dough. Although, it ispreferred to use the method of the present invention for the manufactureof yeast leavened bread products such as bread loaves, rolls or toastbread, the use of the method for any other type of dough and dough basedproducts such as noodle and pasta products and cakes, the quality ofwhich can be improved by the addition of the combination according tothe present invention, is also contemplated.

Preferably the method comprises a further step that the dough is bakedto obtain a baked product.

Preferably, when the dough is a bread dough, the method comprises as afurther step that the dough is baked to obtain a baked product. Oneparticularly desired property of baked bread products is a high specificvolume as defined in the examples. Accordingly, the addition of thecombination of the invention preferably results in an increase of thespecific volume of the baked product that is at least 10%, relative to abaked product made under identical conditions except that the enzyme isnot added. More preferably, the increase of the specific volume is atleast 20% such as at least 30%, e.g. at least 40%. Alternatively, thedough is a dough selected from the group consisting of a pasta dough, anoodle dough, and a cake dough or batter.

The phrase “quality of the product” as used herein refers to the finaland stable volume and/or crust colour and/or texture and taste.

The term “product made from dough” as used herein refers to a breadproduct such as in the form of loaves or rolls, french baguette typebread, pita bread, tacos and crisp bread. Preferably the term refers tocakes, pan-cakes, biscuits. More preferably the term refers to pasta.More preferably the term refers to noodles. More preferably the termrefers to alimentary paste product.

In a preferred embodiment, the oxidoreductase is hexose oxidase. Thefurther ingredients or additive can be any of the ingredients oradditives which are described above. The composition may conveniently bea liquid preparation comprising the oxidoreductase. However, thecomposition is conveniently in the form of dry composition. It will beunderstood that the amount of oxidoreductase activity in the compositionwill depend on the types and amounts of the further ingredients oradditives. However, the amount of oxidoreductase activity is preferablyin the range of 10 to 100,000 units, preferably in the range of 100 to50,000 units such as 1,000 to 10,000 units including 2,000 to 5,000units.

Optionally, the composition may be in the form of a complete doughadditive mixture or pre-mixture for a making a particular finishedproduct and containing all of the dry ingredients and additives for sucha dough. In specific embodiments, the composition may be oneparticularly useful for preparing a baking product or in the making of anoodle product or an alimentary paste product.

As mentioned above, the present invention provides a method forpreparing a bakery product including the addition to the dough of anoxidoreductase such as e.g. hexose oxidase. In particular, this methodresults in bakery products such as the above mentioned products in whichthe specific volume is increased relative to an otherwise similar bakeryproduct, prepared from a dough not containing oxidoreductase. It hasbeen found that the addition of the composition of the present inventionto bakery product doughs results in bakery products such as yeastleavened and chemically leavened products in which the specific volumeis increased relative to an otherwise similar bakery product. In thiscontext, the expression “specific volume” is used to indicate the ratiobetween volume and weight of the product. It has been found that, inaccordance with the method described herein, the specific volume can beincreased significantly such as by at least 10%, preferably by at least20%, including by at least 30%, preferably by at least 40% and morepreferably by at least 50%.

The present invention is highly suitable for improving the rheologicaland/or machineability properties and/or quality (e.g. volume) of thefinished products (products made from the dough) of conventional typesof yeast leavened bread products based on wheat Hour, such as loaves androlls. The present invention is also suitable for improving therheological properties of doughs containing chemical leavening agents(baking powder) and the quality (e.g. volume) of products made from suchdoughs. Such product include as examples breads, sponge cakes andmuffins.

Enzyme Amount

Preferably the or each enzyme is added in an amount from 1-1000 ppm,preferably 25-500 ppm, more preferably 50-300 ppm.

Nucleotide Sequence

The enzyme need not be a native enzyme. In this regard, the term “nativeenzyme” means an entire enzyme that is in its native environment andwhen it has been expressed by its native nucleotide sequence.

The nucleotide sequence of the present invention may be prepared usingrecombinant DNA techniques (i.e. recombinant DNA). However, in analternative embodiment of the invention, the nucleotide sequence couldbe synthesised, in whole or in part, using chemical methods well knownin the art (see Caruthers M H et al (1980) Nuc Acids Res Symp Ser 215-23and Horn T et al (1980) Nuc Acids Res Symp Ser 225-232).

Amino Acid Sequences

The enzyme may be prepared/isolated from a suitable source, or it may bemade synthetically or it may be prepared by use of recombinant DNAtechniques.

Variants/Homologues/Derivatives

The present invention also encompasses the use of variants, homologuesand derivatives of any amino acid sequence of an enzyme of the presentinvention or of any nucleotide sequence encoding such an enzyme. Here,the term “homologue” means an entity having a certain homology with thesubject amino acid sequences and the subject nucleotide sequences. Here,the term “homology” can be equated with “identity”.

In the present context, an homologous sequence is taken to include anamino acid sequence which may be at least 75, 85 or 90% identical,preferably at least 95 or 98% identical to the subject sequence.Typically, the homologues will comprise the same active sites etc. asthe subject amino acid sequence. Although homology can also beconsidered in terms of similarity (i.e. amino acid residues havingsimilar chemical properties/functions), in the context of the presentinvention it is preferred to express homology in terms of sequenceidentity.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

The invention will now be described by way of illustration in thefollowing non-limiting examples and the drawings, in which:

FIG. 1 which is a photographic image of a bread;

FIG. 2 which is a photographic image of a bread; and

FIG. 3 which is a photographic image of a bread.

EXAMPLE 1

1.1. Purification of Hexose Oxidase from Chondrus crispus

A purified hexose oxidase preparation was obtained using the belowextraction and purification procedures. During these procedures and thefollowing characterizations of the purified enzyme, the following assayfor determination of hexose oxidase activity was used:

1.1.1. Assay of Hexose Oxidase Activity

The assay was based on the method described by Sullivan and Ikawa(Biochimica et Biophysica Acta, 1973, 309:11-22), but modified to run inmicrotiter plates. An assay mixture contained 150 μl β-D-glucose (0.1 Min 0.1 M sodium phosphate buffer, pH 6.3), 120 μl 0.1 M sodium phosphatebuffer, pH 6.3, 10 μl o-dianisidinedihydrochloride (Sigma D-3252, 3.0mg/ml in H₂O), 10 μl peroxidase (POD) (Sigma P-8125, 0.1 ml in 0.1 Msodium phosphate buffer, pH 6.3) and 10 μl enzyme (HOX) solution. Blankswere made by adding buffer in place of enzyme solution.

The incubation was started by the addition of glucose. After 15 minutesof incubation at 25° C. the absorbance at 405 nm was read in an ELISAreader. A standard curve was constructed using varying concentrations ofH₂O₂ in place of the enzyme solution.

The reaction can be described in the following manner:

HOX

β-D-glucose+H₂O+O2->gluconic acid+H₂O₂

H₂O₂+β-dianisidine_(red)->2H₂O+o-dianisidine_(ox)

Oxidized o-dianisidine has a yellow colour absorbing at 405 nm.

1.1.2. Extraction

Fresh Chondrus crispus fronds were harvested along the coast ofBrittany, France. This fresh material was homogenized in a pin mill(Alpine). To a 100 g sample of the resulting homogenized frond materialwas added 300 ml of 0.1 M sodium phosphate buffer, pH 6.8. The mixturewas subsequently sonicated in a sonication bath for 5 minutes and thenextracted under constant rotation for 4 days at 5° C., followed bycentrifugation of the mixture at 47,000×g for 20 minutes.

300 ml of the resulting clear pink supernatant was desalted byultrafiltration using an Amicon ultrafiltration unit equipped with anOmega (10 kD cut off, Filtron) ultrafiltration membrane.

1.1.3. Anion Exchange Step

The retentate resulting from 1.1.2 was applied to a 5×10 cm column with200 ml Q-Sepharose FF equilibrated in 20 mM triethanolamine, pH 7.3. Thecolumn was washed with the equilibration buffer and hexose oxidaseeluted with a 450 ml gradient of 0 to 1 M of NaCl in equilibrationbuffer. The column was eluted at 6 ml/minute, and fractions of 14 mlcollected. Fractions 9-17 (total 125 ml) were pooled and concentrated byultrafiltration using an Amicon 8400 unit equipped with an Omega (10 kDcut off, Filtron) ultrafiltration membrane to 7.5 ml.

1.1.4. Gel Filtration

The above 7.5 ml retentate was applied to a Superdex 200 2.6×60 cm gelfiltration column equilibrated in 50 mM sodium phosphate buffer, pH 6.4and eluted at a flow rate of 1 ml/-minute. Fractions of 4 ml werecollected. Fractions 17-28 (total volume 50 ml) containing the hexoseoxidase activity were pooled.

1.1.5. Hydrophobic Interaction Chromatography

To the pool resulting from the gel filtration step 1.1.4 ammoniumsulphate was added to a final concentration of 2 M. This mixture wasthen applied to a 1.6×16 cm column with 32 ml phenyl sepharoseequilibrated in 20 mM sodium phosphate buffer, pH 6.3 and 2 M (NH₄)₂SO₄.The column was washed with equilibration buffer followed by elution ofhexose oxidase at a flow rate of 2 ml/minute using a 140 linear gradientfrom 2 M to 0 M (NH₄)₂SO₄ in 20 mM sodium phosphate buffer. Fractions of4 ml were collected and fractions 24-33 containing the hexose oxidaseactivity were pooled.

The above mentioned pink colour accompanies the enzyme, but it isseparated from hexose oxidase in this purification step.

1.1.6. Mono Q Anion Exchange

The above pool resulting from the above phenyl sepharose chromatographystep was desalted by ultrafiltration as described above. 2 ml of thispool was applied to a Mono Q HR 5/5 column equilibrated in 20 mMtriethanolamine, pH 7.3. The column was subsequently eluted using a 45ml linear gradient from 0 to 0.65 M NaCl in equilibration buffer at aflow rate 1.5 ml/minute. Fractions of 1.5 ml were collected andfractions 14-24 were pooled.

1.1.7. Mono P Anion Exchange

The hexose oxidase-containing pool from the above step 1.1.6 was appliedto a Mono P HR 5/5 column equilibrated in 20 mM bis-Tris buffer, pH 6.5.The enzyme was eluted using a 45 ml linear gradient from 0 to 0.65 MNaCl in equilibration buffer at a flow rate of 1.5 ml/minute, andfractions of 0.75 ml were collected. The highest hexose oxidase activitywas found in fraction 12.

1.2. Characterization of the Purified Hexose Oxidase

The hexose oxidase-containing pools from the above steps 1.1.6 and 1.1.7were used in the below characterization experiments:

1.2.1. Determination of Molecular Weight

The size of the purified native hexose oxidase was determined by gelpermeation chromatography using a Superose 6 HR 10/30 column at a flowrate of 0.5 ml/minute in 50 mM sodium phosphate buffer, pH 6.4. Ferritin(440 kD), catalase (232 kD), aldolase (158 kD), bovine serum albumin (67kD) and chymotrypsinogen (25 kD) were used as size standards. Themolecular weight of the purified hexose oxidase was determined to be120+10 kD.

1.2.2. Determination of pH Optimum

Assay mixtures for the determination of pH optimum (final volume 300 μl)contained 120 μl of 0.1 M stock solution of sodium phosphate/citratebuffer of varying pH values. All other assay mixture components weredissolved in H₂O. The pH was determined in the diluted stock buffersolutions at 25° C. The hexose oxidase showed enzymatic activity from pH3 to pH 8, but with optimum in the range of 3.5 to 5.5.

1.2.3. K_(m) of the Hexose Oxidase for Glucose and Maltose Respectively

Kinetic data were fitted to V=V_(max)S/(Km+S), where V_(max) is themaximum velocity, S is the substrate concentration and Km is theconcentration giving 50% of the maximum rate (Michaelis constant) usingthe EZ-FIT curve fitting microcomputer programme (Perrella, F. W., 1988,Analytical Biochemistry, 174:437-447).

A typical hyperbolic saturation curve was obtained for the enzymeactivity as a function of glucose and maltose, respectively. K_(m) forglucose was calculated to be 2.7 mM±0.7 mM and fur maltose the K_(m) wasfound to be 43.7±5.6 mM.

EXAMPLE 2 Dough Improving Effect of Hexose Oxidase Extracted fromChondrus crispus

2.1. Purification of Hexose Oxidase from Chondrus crispus

For this experiment, hexose oxidase was prepared in the followingmanner:

Fresh Chondrus crispus material was collected at the coast of Brittany,France. The material was freeze-dried and subsequently ground. 40 g ofthis ground material was suspended in 1000 ml of 20 mM triethanolamine(TEA) buffer, pH 7.3 and left to stand at 5° C. for about 64 hours withgentle agitation and then centrifuged at 2000×g for 10 minutes. Thesupernatant was filtered through GF/A and GF/C glass filters followed byfiltering through a 45 μm pore size filter to obtain a filtratepreparation of 800 ml having hexose oxidase activity corresponding to aglucose oxidase activity of 0.44 units per g of preparation. Theactivity was determined using the below procedure.

The supernatant was applied onto a 330 ml bed volume chromatographiccolumn with anionic exchange Q Sepharose Big Beads (dead volume 120 ml).The bound proteins were eluted over 180 minutes using a gradient from 0to 0.5 M NaCl in 20 mM TEA buffer, pH 7.3 followed by 1 M NaCl in 20 mMTEA buffer, and fractions of 9 ml were collected and analyzed for hexoseoxidase activity using the below analytical procedure.

Hexose oxidase activity-containing fractions 60-83 were pooled (about250 ml) and concentrated and desalted by ultrafiltration to about 25 ml.This step was repeated twice on the retentates to which was added 100 ml0.05 mM TEA. The resulting retentate of 25 ml contained 0.95 glucoseoxidase activity units per g.

2.2. Determination of Glucose Oxidase Activity

Definition: 1 glucose oxidase (GOD) unit corresponds to the amount ofenzyme which under the specified conditions results in the conversion of1 μmole glucose per min. The activity is stated as units per g of enzymepreparation.

Reagents: (i) Buffer: 20 g Na₂HPO₄-2H₂O is dissolved in 900 ml distilledwater, pH is adjusted to 6.5; (ii) dye reagent (stock solution): 200 mgof 2,6-dichloro-phenol-indophenol, Sigma No. D-1878 is dissolved in 1000ml distilled water under vigorous agitation for 1 hour; (iii) peroxidase(stock solution): Boehringer Mannheim No. 127 361, 10,000 units isdissolved in 10 ml distilled water and 4.2 g of ammonium sulphate added;(iv) substrate: 10% w/v D-glucose solution in buffer, (v) standardenzyme: hydrase #1423 from Amano.

Analytical principle and procedure: Glucose is converted to gluconicacid and H₂O₂ which is subsequently converted by peroxidase to H₂O andO₂. The generated oxygen oxidizes the blue dye reagent2,6-dichloro-phenol-indophenol which thereby changes its colour topurple. The oxidized colour is measured spectrophotometrically at 590 nmand the enzymatic activity values calculated relative to a standard.

2.3. The Effect of the Hexose Oxidase Preparation on Crosslinkingbetween Thiol Groups in a Wheat Flour Based Dough

The effect of hexose oxidase on the formation of thiol groupcross-linking was studied by measuring the content of free thiol groupsin a dough prepared from 1500 g of wheat flour, 400 Brabender Units (BU)of water, 90 g of yeast, 20 g of sucrose and 20 g of salt to which wasadded 0, 100, 250, 875 and 1250 units per kg of flour, respectively ofthe above hexose oxidase preparation. The measurement was carried outessentially in accordance with the colorimetric method of Ellman (1958)as also described in Cereal Chemistry, 1983, 70, 22-26. This method isbased on the principle that 5.5′-dithio-bis(2-nitrobenzoic acid) (DTNB)reacts with thiol groups in the dough to form a highly coloured anion of2-nitro-5-mercapto-benzoic acid, which is measuredspectrophotometrically at 412 nm.

Assuming that the relative change of the amount of thiol groups in adough is reflected as the change in the optical density (OD) resultingfrom the reaction between thiol groups and DTNB in the dough, thefollowing results were obtained:

Hexose oxidase GOD units/kg flour OD₄₁₂ 0 0.297 100 0.285 250 0.265 8750.187 1250 0.138

Thus, this experiment showed a significant decrease in OD indicating areduction of the content of free thiol groups which was proportionate tothe amount of hexose oxidase activity added.

2.4. Improvement of the Rheological Characteristics of Dough by theAddition of Hexose Oxidase

The above dough was subjected to extensigraph measurements according toAACC Method 54-10 with and without the addition of an amount of thehexose oxidase preparation corresponding to 100 units/kg flour of hexoseoxidase activity. The dough without addition of enzyme served as acontrol.

The principle of the above method is that the dough after forming issubjected to a load-extension test after resting at 30° C. for 45, 90,135 and 180 minutes, respectively, using an extensigraph capable ofrecording a load-extension curve (extensigram) which is an indication ofthe doughs resistance to physical deformation when stretched. From thiscurve, the resistance to extension, B (height of curve) and theextensibility, C (total length of curve) can be calculated. The B/Cratio (D) is an indication of the baking strength of the flour dough.

The results of the experiment is summarized in Table 2.1 below.

TABLE 2.1 Extensigraph measurements of dough supplemented with 100 GODunits/kq flour of hexose oxidase (HOX). Sample Time, min B C D = B/CControl 45 230 180 1.3 HOX 45 320 180 1.8 Control 90 290 161 1.8 HOX 90450 148 3.0 Control 135 290 167 1.7 HOX 135 490 146 3.4 Control 180 300168 1.8 HOX 180 500 154 3.2

It is apparent from this table that the addition of hexose oxidase (HOX)has an improving effect on the doughs resistance to extension asindicated by the increase in B-values. This is reflected in almost adoubling of the B/C ratio as a clear indication that the baking strengthof the flour is significantly enhanced by the hexose oxidase addition.

In a similar experiment, 100 units/kg flour of a commercial glucoseoxidase product was added and the above parameters measured in the samemanner using a dough without enzyme addition as a control. The resultsof this experiment is shown in Table 2.2 below:

TABLE 2.2 Extensigraph measurements of dough supplemented with 100 GODunits/kg flour of glucose oxidase (GOX). Sample Time, min B C D = B/CControl 45 240 180 1.3 GOX 45 290 170 1.7 Control 90 260 175 1.5 GOX 90360 156 2.3 Control 135 270 171 1.6 GOX 135 420 141 3.0

When the results for the above two experiments are compared with regardto differences between control dough and the hexose oxidase or glucoseoxidase supplemented doughs it appeared that hexose oxidase has astronger strengthening effect than glucose oxidase. Furthermore, the B/Cratio increased more rapidly with hexose oxidase relative to glucoseoxidase which is a clear indication that enhancement of the bakingstrength is being conferred more efficiently by hexose oxidase than byglucose oxidase.

EXAMPLE 3 Dough Improving Effect of Hexose Oxidase Extracted fromChondrus crispus

For this experiment fresh Chondrus crispus seaweed fronds were harvestedalong the coast of Hirsholmene, Denmark. Hexose oxidase was isolatedusing two different extraction procedures, and the materials from bothwere pooled for the below dough improving experiment.

3.1. Purification of Hexose Oxidase from Chondrus crispus I

954 g of the fresh fronds was rinsed in distilled water, dried with atowel and stored in liquid nitrogen. The seaweed was blended using aWaring blender and 1908 ml of 0.1 M sodium phosphate buffer, 1 M Na Cl,pH 6.8 was added to the blended seaweed. The mixture was extracted underconstant stirring for 4 days at 5° C., followed by centrifugation of themixture at 20,000×g for 30 minutes.

The resulting 1910 ml supernatant (351.1 U/ml) was concentrated to 440ml at 40° C. in a Buchi Rotavapor R110. The concentrate was ammoniumsulphate fractionated to 25% The mixture was stirred for 30 minutes andcentrifuged for 20 minutes at 47,000×g. The supernatant (395 ml) wasdialysed overnight against 20 l of 10 mM triethanolamine (TEA) buffer,pH 7.3 to a final volume of 610 ml (367.1 U/ml).

The above 610 ml was applied in two runs to a 2.6×25 cm column with 130ml Q-Sepharose FF equilibrated in 20 mM TEA buffer, pH 7.3. The columnwas washed with the equilibration buffer and the bound proteins wereeluted using 800 ml gra dient from 0 to 0.8 M NaCl in equilibrationbuffer. The column was eluted at 4 ml/minute and fractions of 12 mlcollected. Fractions containing the hexose oxidase activity werecollected and pooled to a final volume of 545 ml (241.4 U/ml).

3.2. Purification of Hexose Oxidase from Chondrus crispus II

1250 g of the fresh fronds was rinsed in distilled water, dried with atowel and stored in liquid nitrogen. The seaweed was blended in a Waringblender followed by the addition of 2500 ml 0.1 M sodium phosphatebuffer, 1 M NaCl. pH 6.8. The mixture was extracted under continuousstirring for 4 days at 5° C. followed by centrifugation at 20,000×g for30 minutes.

The resulting 2200 ml supernatant (332.8 U/ml) was concentrated to 445ml at 40° C. using a Buchi Rotavapor R110. The resulting concentrate wasammonium sulphate fractionated to 25%. The mixture was stirred for 30minutes and centrifuged for 20 minutes at 47,000×g. The precipitate wasdiscarded. The 380 ml supernatant was dialysed overnight against 20 l 10mM TEA buffer, pH 7.3, to a final volume of 850 ml (319.2 U/ml).

The above 850 ml was applied to a 2.6×25 cm column with 130 mlQ-Sepharose FF equilibrated in 20 mM TEA buffer, pH 7.3.

The column was washed with the equilibration buffer and the boundproteins were eluted using 800 ml gradient from 0 to 0.8 M NaCl inequilibration buffer. The column was eluted at 4 ml/minute and fractionsof 12 ml collected. Fractions containing the hexose oxidase activitywere collected and pooled to a final volume of 288 ml.

The retentate from the above step was applied to a 2.6×31 cm column with185 ml metal chelating sepharose FF loaded with Ni²⁺ and equilibrated in50 mM sodium phosphate, 1 M NaCl, pH 7.4. The bound proteins were elutedwith a 740 ml gradient of 0 to 35 mM imidazole, pH 4.7 in equilibrationbuffer. The column was eluted at 2 ml/minute and fractions of 11 ml wascollected. Fractions 41-54 (140 ml, 352.3 U/ml) were pooled. Some hexoseoxidase did run through the column.

3.3. Pooling and Concentrating of Extracts

The run through and the 140 ml from purification II and the 545 ml frompurification I were pooled to a final volume of 1120 ml (303.6 U/ml).The 1120 ml was rotation evaporated into a volume of 210 ml followed bydialysis overnight 30 against 20 l of 10 mM TEA buffer, pH 7.3, to afinal volume of 207 ml (1200.4 U/ml)

3.3.1. Anion Exchange Step

The retentate resulting from the above step was applied to a 2.6×25 cmcolumn with 130 ml Q-sepharose FF equilibrated in 20 mM triethanolamine,pH 7.3. The column was washed with the equilibration buffer and thebound proteins eluted using 800 ml gradient from 0 to 0.8 M NaCl inequilibration buffer. The column was eluted at 4 ml/minute and fractionsof 12 ml collected. Fractions 30-50 containing the hexose oxidaseactivity (260 ml, 764.1 U/ml) were collected and pooled.

3.3.2. Other Enzyme Activity

The above pooled solution was tested for the following enzymatic sideactivities catalase, protease, xylanase, α- and β-amylase and lipase.None of these activities were found in the solution.

3.4. Improvement of the Rheological Characteristics of Dough by theAddition of Hexose Oxidase

A dough was prepared from wheat flour, water and salt and 0, 72, 216 and360 units per kg of flour, respectively of the above hexose oxidasepreparation was added hereto. The dough without addition of enzymeserved as a control. In addition two doughs were prepared to which wasadded 216 and 360 units per kg of flour respectively, of Gluzyme, aglucose oxidase available from Novo Nordisk A/S. Denmark.

The doughs were subjected to extensigraph measurements according to amodification of the above AACC Method 54-10. The results of theexperiment are summarized in Table 3.1 below.

TABLE 3.1 Extensigraph measurements of dough supplemented with hexoseoxidase (HOX) or glucose oxidase (units per kg flour) Sample Time, Min BC D = B/C Control 45 250 158 1.6 HOX 72 U/kg 45 330 156 2.1 HOX 216 U/kg45 460 153 3.0 HOX 360 U/kg 45 580 130 4.5 Gluzyme 72 U/kg 45 350 1592.2 Gluzyme 216 U/kg 45 340 148 2.3 Gluzyme 360 U/kg 45 480 157 3.1Control 90 290 164 1.8 HOX 72 U/kg 90 470 145 3.2 HOX 216 U/kg 90 650142 4.6 HOX 360 U/kg 90 870 116 7.5 Gluzyme 72 U/kg 90 450 147 3.1Gluzyme 216 U/kg 90 480 138 3.5 Gluzyme 360 U/kg 90 500 152 3.2 Control135 330 156 2.1 HOX 72 U/kg 135 540 129 4.2 HOX 216 U/kg 135 750 125 6.0HOX 360 U/kg 135 880 117 7.5 Gluzyme 72 U/kg 135 510 136 3.8 Gluzyme 216U/kg 135 550 122 4.5 Gluzyme 360 U/kg 135 560 121 4.6

It is evident from the above table that the addition of hexose oxidase(HOX) or glucose oxidase had an improving effect on the resistance ofdoughs to extension as indicated by the increase in B-values. This isreflected in an increase of the B/C ratio as a clear indication that thebaking strength of the flour was enhanced significantly by the additionof enzymes.

It is also evident that the hexose oxidase had a higher strengtheningeffect than glucose oxidase. Furthermore, the B/C ratio increased morerapidly with hexose oxidase relative to glucose oxidase which is a clearindication that enhancement of the baking strength is being conferredmore efficiently by hexose oxidase than by glucose oxidase.

EXAMPLE 4 Dough Improving Effect of Hexose Oxidase Extracted fromChondrus crispus

4.1. Purification of Hexose Oxidase from Chondrus crispus

Fresh Chondrus crispus fronds were harvested along the coast ofBrittany, France. 2285 g of this fresh material was rinsed in distilledwater, dried with a towel and stored in liquid nitrogen. The seaweed wasblended in a Waring blender followed by addition of 4570 ml 0.1 M sodiumphosphate buffer, 1 M NaCl pH 6.8. The mixture was extracted undercontinuous magnetic stirring for 4 days at 5° C. followed bycentrifugation at 20,000×g for 30 minutes.

The resulting 4930 ml supernatant (624.4 U/ml) was concentrated to 1508ml at 40° C. using a Buchi Rotavapor R110. The obtained concentrate waspolyethylenglycol fractionated to 3% (w/v). The mixture was stirred for30 minutes and centrifuged for 30 minutes at 47,000×g. The pellet wasdiscarded. The 1470 ml supernatant (2118.7 U/ml) was PEG fractionated to24%. The mixture was stirred for 30 minutes and centrifuged for 30minutes at 47,000×g. The supernatant was discarded and the 414.15 g ofprecipitate was resuspended in 200 ml 20 mM TEA buffer, pH 7.3, followedby dialysis over night at 5° C. against 20 l 10 mM TEA buffer, pH 7.3.

After dialysis the volume was 650 ml (2968.6 U/ml). The suspension wascentrifuged for 30 minutes at 20,000×g. The precipitate was discardedand the supernatant was diluted to 3200 ml with distilled water.

The above 3200 ml (829.9 U/ml) was applied to a 10×14 cm column with1100 ml Q-Sepharose FF equilibrated in 20 mM TEA buffer, pH 7.3. Thecolumn was washed with the equilibration buffer and the bound proteinswere eluted using 15,000 ml gradient from 0 to 0.8 M NaCl inequilibration buffer. The column was eluted at 50 ml/minute. Hexoseoxidase did run through the column and 840 ml of this was collected.

The 840 ml suspension was treated with kieselguhr and concentrated to335 ml (2693.3 U/ml).

The above 335 ml was applied to a 3 l Sephadex G25C desalting column10×40 cm. The column was equilibrated in 20 mM TEA buffer, pH 7.3,eluted at a flow rate of 100 ml/minute and 970 ml eluate was collected.This eluate was applied to a 10×14 cm column with 1100 ml Q-Sepharose FFequilibrated in 20 mM TEA, pH 7.3. The column was washed with theequilibration buffer and bound proteins eluted using a 15,000 mlgradient of 0 to 0.8 M NaCl in equilibration buffer. The column waseluted at 50 ml/min. Hexose oxidase did run through the column and 1035ml of this was collected.

To the above eluate (1035 ml) ammonium sulphate was added to a finalconcentration of 2 M. The mixture was then applied in two runs to a 5×10cm column with 200 ml phenyl sepharose HP equilibrated in 25 mM sodiumphosphate buffer, pH 6.3 and 2 M (NH₄)₂SO₄. The column was washed withequilibration buffer followed by eluting the bound proteins at a flowrate of 50 ml/minute using 5,000 ml gradient from 2 M to 0 M (NH₄)₂SO₄in 25 mM sodium phosphate buffer. Fractions of 500 and 29 ml,respectively were collected from run 1 and 2. Fraction 5 in run 1 andfractions 27-42 in run 2 containing the hexose oxidase activity werepooled to a total of 1050 ml (563.9 U/ml).

The above pool was desalted by a 3 l Sephadex G25C gel filtrationcolumn. The column was equilibrated in 20 mM TEA buffer, pH 7.3, elutedat a flow rate of 100 ml/minute and 1,000 ml eluate was collected.

The 1,000 ml eluate was concentrated to 202 ml (2310.2 U/ml) and thispreparation was used for following rheology testing.

4.2. Improvement of the Rheological Characteristics of Dough by theAddition of Hexose Oxidase

A dough was prepared from wheat flour, water and salt and 0, 288, 504and 720 oxidoreductase units per kg of flour, respectively of the abovehexose oxidase preparation was added hereto. The dough without additionof enzyme served as a control. In addition two doughs were prepared towhich was added 288 and 504 oxidoreductase units per kg of flourrespectively, of Gluzyme, a glucose oxidase available from Novo NordiskA/S. Denmark.

The doughs were subjected to extensigraph measurements according to amodification of AACC Method 54-10.

The results of the experiment are summarized in Table 4.1 below.

TABLE 4.1 Extensigraph measurements of dough supplemented with hexoseoxidase (HOX) or glucose oxidase (Units per kg flour). Sample Time, MinB C D = B/C Control 45 210 171 1.2 HOX 288 U/kg 45 490 139 3.5 HOX 504U/kg 45 640 122 5.2 HOX 720 U/kg 45 730 109 6.7 Gluzyme 288 U/kg 45 350165 2.1 Gluzyme 504 U/kg 45 385 153 2.5 Gluzyme 720 U/kg 45 435 148 2.9Control 90 275 182 1.5 HOX 288 U/kg 90 710 130 5.5 HOX 504 U/kg 90 825106 7.8 HOX 720 U/kg 90 905 107 8.5 Gluzyme 288 U/kg 90 465 153 3.0Gluzyme 504 U/kg 90 515 135 3.8 Gluzyme 720 U/kg 90 540 140 3.9 Control135 280 175 1.6 HOX 288 U/kg 135 745 102 7.3 HOX 504 U/kg 135 920 94 9.8HOX 720 U/kg 135 — 80 — Gluzyme 288 U/kg 135 525 129 4.1 Gluzyme 504U/kg 135 595 129 4.6 Gluzyme 720 U/kg 135 630 121 5.2

It is apparent from the above results that the addition of hexoseoxidase (HOX) or glucose oxidase has an improving effect on theresistance of doughs to extension as indicated by the increase inB-values. This is reflected in an increase of the B/C ratio.

It is also apparent that hexose oxidase has a stronger s strengtheningeffect than that of glucose oxidase, the strengthening effect of bothenzymes being proportional to the amount of enzyme added. Furthermore,the B/C ratio increased more rapidly with hexose oxidase relative toglucose oxidase which is a clear indication that enhancement of thebaking strength is being conferred more efficiently by hexose oxidasethan by glucose oxidase.

EXAMPLE 5 Improving Effect of Hexose Oxidase Extracted from Chondruscrispus on the Specific Volume of Bread

5.1. Purification of Hexose Oxidase from Chondrus crispus

Fresh Chondrus crispus fronds were harvested along the coast ofBrittany, France. 2191 g of this fresh material was rinsed in distilledwater, dried with a towel and stored in liquid nitrogen. The seaweed wasblended in a Waring blender fol lowed by addition of 4382 ml 0.1 Msodium phosphate buffer, 1 M NaCl and pH 6.8. The mixture was extractedunder continuously magnetic stirring for 4 days at 5° C. followed bycentrifugation at 20,000×g for 20 minutes.

The resulting 4600 ml supernatant (746.1 U/ml) was concentrated to 850ml at 40° C. in a Buchi Rotavapor R110. This concentrate (3626.9 U/ml)was polyethylene glycol fractionated to 3% (w/v). The mixture wasstirred for 30 minutes and centrifuged for 30 minutes at 20,000×g. Theprecipitate was discarded. The 705 ml supernatant (2489.8 U/ml) was PEGfractionated to 25%. The mixture was stirred for 30 minutes andcentrifuged for 30 minutes at 20,000×g. The supernatant was discardedand the 341 g of precipitate was resuspended in 225 ml 20 mM TEA buffer,pH 7.3. The suspension (500 ml) was desalted on a 3 l Sephadex G25Cdesalting column 10×40 cm. The column was equilibrated in 20 mM TEAbuffer, pH 7.3, and eluted at a flow rate of 100 ml/minute. 1605 mleluate was collected.

To the above eluate (687.5 U/ml) ammonium sulphate was added to a finalconcentration of 2M. The mixture was then applied in two runs to a 5×10cm column with 200 ml phenyl sepharose HP equilibrated in 25 mM sodiumphosphate buffer, pH 6.3 and 2 M (NH₄)₂SO₄. The column was washed withequilibration buffer followed by elution of the bound proteins at a flowrate of 50 ml/minute using 5,000 ml gradient from 2 M to 0 M (NH₄)₂SO₄in 25 mM sodium phosphate buffer. Fractions of 29 ml was collected.Fractions 85-105 in run 1 and fractions 36-69 in run 2 containing thehexose activity were pooled to a total of 1485 ml (194.7 U/ml).

The above pool was desalted by a 3 l Sephadex G25C gelfiltration column,the same as used in 4.1. The column was equilibrated in 20 mM TEAbuffer, pH 7.3, and eluted at a flow rate of 100 ml/minute. 1,200 mleluate was collected.

The 1,200 ml eluate was concentrated to 685 ml (726.2 U/ml) and used forbaking experiments.

5.2. Improvement of the Specific Volume of Bread by Adding HexoseOxidase to the Dough

A dough was prepared from 1500 g of flour, 90 g of yeast, 24 g of salt,24 g of sugar and 400 BU of water and 0 or 108 units of the abovepurified hexose oxidase and 108 units of Gluzyme (glucose oxidaseavailable from Novo Nordisk, Denmark) per kg flour, respectively wasadded hereto. The dough was mixed on a Hobart mixer for 2+9 minutes at26° C. and divided into two parts followed by resting for 10 minutes at30° C. in a heating cabinet, moulding with a Fortuna 3/17/7 and proofingfor 45 minutes at 34° C. and 85% RH. The thus proofed dough was baked at220° C. for 17 minutes with 12 sec. steam in a Bago oven.

The results of the experiment are summarized in table 5.1 below.

TABLE 5.1 Improvement of specific volumes of bread prepared from doughsupplemented with hexose oxidase or glucose oxidase (Units per kg flour)Total volume Total weight Specific volume control 5325 1027 5.18 Hexoseoxidase 108 U/kg 6650 1036 6.41 Gluzyme 108 U/kg 6075 1030 5.89

It is evident from the above table that the addition of hexose oxidaseor glucose oxidase had an increasing effect on the total volume, theweight being essentially the same. This is reflected in an increase ofthe specific volume as compared to the bread baked without addition ofenzymes.

It is also evident that hexose oxidase has a significantly larger effecton the increase of the specific volume than had glucose oxidase at thesame dosage.

EXAMPLE 6 Characterization of the Purified Hexose Oxidase

Preparations from the above purifications were used for characterizationof hexose oxidase.

6.1. Staining for Hexose Activity After Non-Denaturing PAGE

Hexose oxidase activity was analyzed by native PAGE using precast 8-16%Tris-glycine Novex gels according to the manufactures instructions(Novex, San Diego, USA). After electrophoresis the gels were stained forhexose oxidase activity by incubation of the gel in a solutioncontaining 50 mM sodium phosphate buffer, pH 6.0, 100 mM glucose, 50mg/l phenazine methosulphate (Sigma P9625) and 250 mg/l nitrobluetetrazolium (Sigma N6876) as described in the PhD thesis by Witteveen,C. F. B. (1993) “Gluconate formation and polyol metabolism inAspergillus niger”. After about 30 minutes the hexose oxidase activitywas visible as a double hand very close to each other. The same doubleband was also seen when a native PAGE of hexose oxidase was silverstained. The molecular weight of purified hexose oxidase was determinedto 144 kD by native PAGE. Half the gel was silver stained, the otherhalf was activity stained. As standards were used bovine serum albumin(67 kD), lactate dehydrogenase (140 kD), catalase (232 kD), ferritin(440 kD) and thyroglobulin (669 kD).

6.2 Determination of Molecular Weight by SDS-Page

The molecular weight was also determined on material which was firstapplied to a native PAGE as described above, after activity staining thehexose oxidase band was excised from the gel and then electroelutedusing an Electro-Eluter (model 422, Bio-Rad, CA, USA) according to themanufacturer's recommendations. The electroeluted protein was subjectedto SDSPAGE and silver stained. This material gave “one” double band atabout 70 kDa in SDS-PAGE gels. The electroeluted hexose oxidase istherefore a dimer of two subunits.

6.3 Determination of pI of Hexose Oxidase

Samples containing hexose oxidase activity were analyzed by isoelectricfocusing (IEF) using a precast 3-10 IEF gel according to themanufacturer's recommendations (Novex, San Diego, US). Afterelectrophoresis half of the gel was silver stained and the other halfnitroblue tetrazolium stained as described in 6.1.

Hexose oxidase stained as a double band. The pI of the first band was4.79, pI of the second band was 4.64. As standards were used trypsinogen(9.30), lentil lectin basic band (8.65), lentil lectin middle band(8.45), lentil lectin acid band (8.15), horse myoglobin acidic band(6.85), human carbonic anhydrase B (5.85), β-lactoglobulin A (5.20), soybean trypsin inhibitor (4.55) and amyloglucosidase (3.50).

6.4 Determination of Km Hexose Oxidase for Different Sugars

Km of hexose oxidase was determined for 7 different sugars as describedin 1.2.3. Results are summarized in table 6.1 below.

TABLE 6.1 Determination of Km of hexose oxidase for different sugarsSubstrate Km (mM) cv (mM) D-glucose 2.7 0.7 D-galactose 3.6 1 cellobiose20.2 7.8 maltose 43.7 5.6 lactose 90.3 20.6 xylose 102 26 arabinose 531158 (cv = coefficient of variation)6.5 Determination of a Peptide Sequence of Hexose Oxidase

50 μl from the electroeluted mixture in 6.2 was suspended in 450 μl 0.1%triflouracetic acid (TFA).

To remove the Tris, glycine and SDS, the above mixture was subjected tochromatography on reverse-phase HPLC. The resulting solution was appliedin 9 runs to a 4.6×30 cm Brownlee C2 column equilibrated in 0.1% TFA.The column was washed in equilibration buffer and bound peptides elutedwith a 14 ml gradient from 10 to 80% acetonitrile in 0.1% TFA, at a flowrate of 0.7 ml/min. Fractions from the largest peak containing theenzyme were collected and freeze dried.

6.5.1 Endoproteinase Lys-C Digestion

The resulting freeze dried enzyme was dissolved in 50 μl 8 M urea, 0.4 MNH₄HCO₃, pH 8.4. Denaturation and reduction of the protein was carriedout by the addition of 5 μl 45 mM di-thiothreitol and under an overlayof N₂ at 50° C. for 15 min. The solution was cooled to room temperatureand 5 μl 100 mM iodoacetamide was added, the cysteines being derivatizedfor 15 min. at room temperature in the dark under N₂. Subsequently, thesolution was suspended in 135 μl water and digestion was carried out at37° C. under N₂ for 24 hours by addition of 5 μg endoproteinase Lys-Cdissolved in 5 l water. The reaction was terminated by freezing thereaction mixture at −20° C.

6.5.2 Reverse-Phase HPLC Separation of Peptides

The resulting peptides were separated by reverse-phase HPLC on a VYDACc18 column 0.46×15 cm (The Separation Group, CA, USA) using as solvent A0.1% TFA in water and as solvent B 0.1% TFA in acetonitrile.

6.5.3 Peptide Sequencing

Sequencing was performed on an Applied Biosystems 476A sequencer(Applied Biosystems, CA, USA) using pulsed-liquid fast cycles accordingto the manufacturer's instructions. A peptide having the below aminoacid sequence was identified:

D P C Y I V I D V N A G T P O K P D P.

EXAMPLES 7 TO 10

Definitions

All PANODAN™ products contain DATEM (Di-acetyl tartaric acid ester ofmonoglycerides) and are obtained from Danisco A/S.

PANODAN™ 521: DATEM containing bacterial xylanase and fungal amylase

TS-E 662™ (obtained from Danisco A/S) is a product containing hexoseoxidase (Hox) (EC 1.1.1.5) from Chondrus chrispus expressed in Hansenulapolymorpha.

TS-E 680™ (obtained from Danisco A/S) is a product containing fungalxylanase (EC 3.2.1.8) from Aspergillus niger.

TS-E 861™ (obtained from Danisco A/S) is a product containing fungalxylanase (EC 3.2.1.8) from Aspergillus niger, lipase (EC 3.1.1.3) fromThermomyces lanuginosa expressed in Aspergillus oryzae, and hexoseoxidase (EC 1.1.1.5) from Chondrus crispus expressed in Hansenulapolymorepha.

GRINDAMYL™ H 640 (obtained from Danisco A/S): contains bacterialxylanase

Grindamyl™ H 121 (obtained from Danisco A/S) is a fungal xylanase (EC3.2.1.8) from Aspergillus niger.

Grindamyl™ EXEL 16 (obtained from Danisco A/S) is lipase (EC 3.1.1.3)from Thermomyces lanuginosa expressed in Aspergillus oryzae.

Grindamyl™ EXEL 66 (obtained from Danisco A/S) is a mixture of lipase(EC 3.1.1.3) from Thermomyces lanuginosa expressed in Aspergillus oryzaeand a fungal xylanase (EC 3.2.1.8) from Aspergillus niger.

Lipopan F™ (Lipopan F BG) (obtained from Novozymes) is according to itsproducer (Novozymes) a purified lipolytic enzyme from Fusarium oxysporumproduced by submerged fermentation of a genetically modified Aspergillusoryzae microorganism. According to its producer, Lipopan F has inherentactivity toward phospholipids, glycolipids and triglycerides.

Recipes/Procedures

High Volume Tweedy

Recipe

Product Name % Gram ppm Ijsvogel flour 3000 Water 58 Salt 60 Compressedyeast 180 Ascorbic acid 30Procedure:

-   -   Dough temperature: 29° C. (dough temp.-flour temp.+4° C. water        temp.)    -   Mixing: 55 WH no vacuum    -   Resting: 5 min. at room temperature    -   Scaling: 500 g (bread), 1350 g (rolls)    -   Resting: 5 min. at room temperature    -   Moulding: Puma I 13 II 18 (bread), Fortuna 3/17/7 (rolls),        Glimek (moulding machine) 1:4, 2:3, 3:12, 4:14    -   Proofing: 70 min. at 43° C., 70% RH. (bread), 50 min. at 34° C.,        85% RH. (rolls)    -   Baking: BAGO, 35 min.+5 min. with the steamer open at 220° C.,        12 sec. steam (bread), 17 min. at 220° C., 17 sec. steam (rolls)        Turkish Batard        Recipe

Product name % Gram ppm Ijsvogel flour 2000 Water 57,00 Compressed yeast80 Salt 30 Ascorbic acid 70Procedure:

-   -   Flour temperature: 15-17° C. (for trials—storage day before use        at 15° C.)    -   Mixing: 35 min. After 25 min. acid salt    -   After 30 min. add yeast    -   Dough temp.: 23-25° C.    -   Resting: 30 min. Bulk rest on table (table=22° C. & 80% RH)    -   Scaling 300 g. pieces    -   Rounding: By hand    -   Resting: 25 min. on table (table=22° C. & 80% RH) . . . start        clock when scaling starts    -   Molding=Glimek: 1:5, 2:4, 3:15, 4:10 . . . 10 in innerpos.    -   Proofing: 60 min & 90 min. for this trial at 30° C. & 85% RH    -   Shock test    -   Baking: 20 min. in Bago1 & 25 min. in Bago2 . . . the last 5        min. is with the damper open for both ovens.    -   Bago1: 250° C. start temp. 5 sec. steam with damper open. Oven        temp. clown to 230° C. at once. Close damper after 11% min.    -   Bago2: 275° C. start temp. 8 sec. steam with damper open. Oven        temp. down to 260° C. at once. Close damper after 11% min.        Crispy Rolls        Recipe:

Product Name % Gram ppm Danish silver flour 2000 Water 58/60 Compressedyeast 120 Salt 32 Sugar 32 Ascorbic acid 40Procedure:

-   -   Mixing: Diosna 2+5 min. (depending on flour)    -   Dough temperature: 26° C.    -   Scaling: 1350 g    -   Resting: 10 min. at 30° C. in heating cabinet    -   Moulding: Fortuna 3/17/7    -   Proofing: 45 min alternatively 90 min at 34° C., 85% RH.    -   Baking: 18 min. at 220° C., 8 sec. steam (Bago-oven), 7 sec.        steam (Wachtel-oven)    -   (MIWE program 28) (0.35 liter steam, 15 min. at 2000° C., ½ min.        at 2200° C.)        US Toast

Here a sponge as a pre-mix is prepared, to all of which is then addedthe dough.

Recipe Gr % US Flour 900.000 g 50.000% Sponge: Water 900.000 g 50.000%Dry 23.400 g  1.300% Yeast Yeast Food 5.400 g  0.300% Enzyme 0.054 g 0.003% Complex ADA 0.036 g  0.002% US Flour 900.000 g 50.000% Dough:Water 234.000 g 13.000% Dry 25.200 g  1.400% Yeast Sugar 153.000 g 8.500% Salt 43.200 g  2.400% Shortening (fat) 36.000 g  2.000%Sod.Prop. 8.100 g  0.450% Dimodan SDM-T (P100/B) 9.000 g  0.500% Asc.Acid. 0.072 g  0.004% ′→ (=7,200 g to 1000 ml. Take 10 ml. from thesolution) Total flour amount: 1.800,000 g. Datem 22-CA-60 4.500 g0.2500% S685 300 PPM H640 20 PPM TS-E 662 100 PPM

-   -   Care has to be taken with the water amount added from asc. acid        solution and other water based solutions ex. enzymes.    -   The extra added water amount should be be withdrawn from the        water amount on the Dough-side of the recipe.

The enzyme complex is a mix of alpha amylase and amyloclucosidase.

DIMODAN SDM-T (P100/B) (obtained from Danisco A/S) is a distilledmonoglyceride.

Procedure:

For the Sponge: Water Temp.: 25° C. Hobart mixer Step 1, 1 min. Step 2,1 min. Step 3, 1 min.

Fermentation: 2 h & 15 min. 40° C. & 80% RH (relative humidity) 45 min.in freezer.

-   -   For the Dough:    -   Mix all ingredients together    -   Diosna-Mixer: Speed 1, 120 secs & Speed 2, 450 secs (or 28        degrees dough temp.)    -   On table—rest 5 min.    -   Weigh out the breads at 450 g pr. bread—rest 5 min.

Glimek (moulding machine) adjustments: 1, 2, 14, 11—& 9 cm—read on outerposition.

Fermentation:

-   -   1 h & 10 min. 45 degrees Celsius & 90% RH

Bake-off:

-   -   Start temp.=250 degrees Celsius in 25 min.

Insert the breads and adjust bake-off temperature to 200 degrees Celsiusat once.

Baking Trials

In each trial the dough characteristic, stickiness and all over breadscore have been evaluated. The dough characteristic is a total of threedifferent parameters: dough extensibility evaluated just after mixingand again after resting and stickiness after resting. Each parameter hasbeen evaluated by bakers on a scale from 1-10, where 10 are the best.The score in the examples are a total of these different scores.

Stickiness evaluation has been subjectively evaluated by bakers justafter mixing on a scale from 1 to 10, where 10 is the best, meaning nonsticky.

All over bread score is a total of an evaluation made on bread crust,-crumb, possible capping and all over energy of the bread. Again eachparameter is evaluated on a scale from 1-10, where 10 is the best.

EXAMPLE 7 Testing Alternatives in Tweedy Bread (UK Procedure)

The breads were rested for 70 min each and after a full proofing, eachbread was shock treated in order to evaluate the shock resistance andthereby the dough stability.

In the baking trials, both pure enzyme solutions and combinations ofDATUM and enzymes were tested as alternative to Lipopan F.

Baking Trials 4969-29

All Specific Shocked Dough Dough over volume, volume, charac- sticki-bread Test ccm/g ccm/g teristic ness score 0.4% PANODAN GB 5.6 4.64 15 429 0.2% PANODAN GB, 5.75 4.92 14 4 30 100 ppm GRINDAMYL H121, 100 ppmTS-E 662 100 ppm TS-E 662, 5.57 4.47 14 4 20 100 ppm GRINDAMYL H121, 100ppm GRINDAMYL EXEL 16 40 ppm Lipopan F 5.7 4.6 13 4 29 0.2% PANODAN GB,5.88 4.6 14 4 27 20 ppm Lipopan F 20 ppm Lipopan F, 5.65 4.78 14 4 29100 ppm TS-E 662, 100 ppm GRINDAMYL H121 40 ppm Lipopan F, 5.79 4.82 134 29 100 ppm TS-E 662, 100 ppm GRINDAMYL H121

From the results it can be concluded that PANODAN GB results in a bettercrust of the product and a product.

The combination of PANODAN GB in combination with xylanase and hexoseoxidase yields a beneficial effect.

When using DATEM and/or HOX in combination with GRINDAMYL EXEL 66 thevolume is increased significantly and the crust is considerablyimproved. The test with 0.1% PANODAN GB 100 ppm GRINDAMYL EXEL 66 and100 ppm TS-E 662 (HOX), gave a significantly good result at the samelevel as 0.4% PANODAN GB. Use of DATEM clearly gives a significantlypositive effect on the crust as compared to pure enzyme solutions.

EXAMPLE 8 Testing Alternatives in Turkish Batard

Baking Trials 7258-2

Specific Dough Dough All over volume, characteristic stickiness breadTest ccm/g * ** score*** 15 ppm Lipopan F, 60 5.01 14 4 33 ppm TS-E 68040 ppm Lipopan F 3.78 15 5 32 100 ppm TS-861* 5.03 16 5 44 *Acombination of fungal xylanase. 1,3 triglyceride degrading lipase andhexose oxidase.

Both from the specific volume in the table as well as the pictures shownin FIGS. 1-3 it can be concluded that TS-E 861 performs better.

EXAMPLE 9 Testing Alternatives in Crispy Rolls

The rolls were fermented at two different fermentation times—45 and 90min in order to stress the system and thereby give a better picture ofthe dough strengthening effect of the products. In general it can besaid that 90 min of fermentation for a small crispy roll is quite long.

Baking Test: 4969-28

Specific Specific Dough All over volume volume Dough sticki- bread 45min, 90 min, characteristic ness score Test ccm/g ccm/g * ** *** 0.3%PANODAN 7.15 8.48 14 5 25 A2020 30 ppm Lipopan F 6.83 8.1 14 4 26 100ppm TS-E 662, 6.98 8.98 14 5 27 100 ppm GRINDAMYL H121, 100 ppmGRINDAMYL EXEL 16

From the results it can be seen that use of the combination of xylanase,1,3 triglyceride degrading lipase and hexose oxidase produces beneficialresults.

In short fermentation times (45 min.) at certain concentrations PANODANA2020 and Lipopan F gave comparable volume results. However, 0.3%PANODAN A2020 showed better results with regard to crispiness of thecrust and a better dough stability in general. We found that Lipopan Foften gave a slightly more “wet” crust.

Using HOX in combination with GRINDAMYL EXEL 66 and PANODAN 660 resultsin an increase in dough stability.

With prolonged fermentation times (90 min.) all buns become relativelyunstable. At some concentrations PANODAN A2020 does, however, give thebest result.

EXAMPLE 10 Testing Alternatives in US Toast

Test of Lipopan F in a US sponge and dough using flour from Mexico—hardwheat type. The breads have all been fully proofed and after that eachbread have been shock treated in order to evaluate the shock resistanceand thereby the dough stability.

Baking Trials 7230-1:

Specific Shocked Test volume, ccm/g volume, ccm/g 0.5% PANODAN 521 6.885.47 10 ppm Lipopan F 6.16 5.36 20 ppm Lipopan F 6.44 5.30 40 ppmLipopan F 6.28 5.52 0.25% PANODAN 521, 7.15 5.74 20 ppm GRINDAMYL H 640,100 ppm TS-E 662

From these tests it is clear that the use of Hox results in a far betterdough stability and consequently an increase of volume.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention may be apparent tothose skilled in the art without departing from the scope and spirit ofthe present invention. Although the present invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in biochemistry and biotechnology or related fields areintended to be within the scope of the claims.

Summary Paragraphs

Some aspects of the present invention are now described by way ofSummary Paragraphs.

1. A method of improving the rheological properties of a flour dough andthe quality of the finished product made from the dough, comprisingadding to the dough ingredients, dough additives or the dough aneffective amount of an oxidoreductase which is at least capable ofoxidizing maltose.

2. A method according to paragraph 1 wherein the oxidoreductase ishexose oxidase.

3. A method according to paragraph 2 wherein the hexose oxidase isderived from a source selected from an algal species, a plant speciesand a microbial species.

4. A method according to paragraph 3 wherein the hexose oxidase isderived from Chondrus crispus.

5. A method according to paragraph 2 wherein hexose oxidase is added inan amount which is in the range of 1 to 10,000 units per kg of flour.

6. A method according to paragraph 5 wherein the hexose oxidase is addedin an amount which is in the range of 10 to 1000 units per kg of flour.

7. A method according to paragraph 1 or 2 wherein the resistance toextension of the dough in terms of the ratio between the resistance toextension (height of curve, B) and the extensibility (length of curve,C), i.e. the B/C ratio, as measured by the AACC method 54-10 isincreased by at least 10% relative to that of an otherwise similar doughnot containing oxidoreductase.

8. A method according to paragraph 1 wherein the finished product isbread.

9. A method according to paragraph 1 wherein the finished product is anoodle product

10. A method according to paragraph 1 wherein the finished product is analimentary paste product.

11. A method according to paragraph 1 wherein at least one furtherenzyme is added to the dough ingredients, dough additives or the dough.

12. A method according to paragraph 11 wherein the further enzyme isselected from the group consisting of a cellulase, a hemicellulase, axylanase, a starch degrading enzyme, a glucose oxidase, a lipase and aprotease.

13. A dough improving composition comprising an oxidoreductase which isat least capable of oxidising maltose and at least one further doughingredient or dough additive.

14. A composition according to paragraph 13 wherein the oxidoreductaseis derived from a source selected from an algal species, a plant speciesand a microbial species.

15. A composition according to paragraph 14 wherein the oxidoreductaseis hexose oxidase.

16. A composition according to paragraph 15 wherein the hexose oxidaseis derived from Chondrus crispus.

17. A composition according to paragraph 13 which is a pre-mixtureuseful for preparing a baked product or in making a noodle product or analimentary paste product.

18. A composition according to paragraph 13 which comprises an additivefrom the group consisting of an emulsifying agent and a hydrocolloid.

19. A composition according to paragraph 18 wherein the hydrocolloid isselected from the group consisting of an alginate, a carrageenan, apectin and a vegetable gum.

20. A method of preparing a bakery product the method comprisingpreparing a flour dough to which is added an effective amount of anoxidoreductase which is at least capable of oxidising maltose, andbaking the dough.

21. A method according to paragraph 20 wherein the specific volume ofthe bakery product is increased relative to an otherwise similar bakeryproduct prepared from a dough not containing oxidoreductase.

22. A method according to paragraph 21 wherein the specific volume isincreased by at least 20%.

23. A method according to paragraph 20 wherein at least one furtherenzyme is added to the dough.

24. A method according to paragraph 20 wherein the further enzyme isselected from the group consisting of a cellulase, hemicellulase, axylanase, a starch degrading enzyme, a glucose oxidase, a lipase and aprotease.

25. A method according to paragraph 20 wherein the oxidoreductase ishexose oxidase.

26. A method of preparing a flour dough-based food product, comprisingadding to the dough an effective amount of a maltose oxidisingoxidoreductase.

27. A method according to paragraph 26 wherein the oxidoreductase ishexose oxidase.

1. A method of improving the rheological and/or machineabilityproperties of a flour dough and/or the quality of the product made fromthe dough, comprising adding to the dough a combination comprising ahexose oxidase and an emulsifying agent, wherein the emulsifying agentis a lipase.
 2. A method according to claim 1 wherein the lipasecomprises a triacylglycerol lipase, a galactolipase, or a phospholipase.3. A method according to claim 1 wherein the hexose oxidase is isolatedfrom a red algae.
 4. A method according to claim 1 wherein the flourdough comprises flour, water and at least one further dough additive oringredient.
 5. A method according to claim 1 wherein the flour doughcomprises flour, water and at least one further dough additive oringredient and wherein the further dough additive or ingredient isselected from the group consisting of a vegetable oil, a vegetable fat,an animal fat, shortening, butterfat, glycerol, milk fat and a mixturethereof.
 6. A method according to claim 1 wherein the flour doughcomprises a hard flour.
 7. A method according to claim 1 wherein theproduct is a bread product.
 8. A method according to claim 1 wherein atleast one further enzyme is added to the dough.
 9. A method according toclaim 1 wherein at least one further enzyme is added to the dough, andwherein the further enzyme comprises a xylanase, a cellulase, ahemicellulase, a starch degrading enzyme, a protease, a lipoxygenase, anoxidoreductase or a lipase.
 10. A dough improving composition comprisinga hexose oxidase and an emulsifying agent, wherein the emulsifying agentis a lipase.
 11. A dough improving composition according to claim 10wherein the lipase comprises a triacylglycerol lipase, a galactolipase,or a phospholipase.
 12. A dough improving composition according to claim10 wherein the lipase comprises a triacylglycerol lipase, agalactolipase, or a phospholipase and wherein the hexose oxidase isisolated from red algae.
 13. A dough improving composition according toclaim 10 wherein the dough improving composition comprises at least onefurther dough additive or ingredient.
 14. A dough improving compositionaccording to claim 10 wherein the dough improving composition comprisesat least one further dough additive or ingredient and wherein thefurther dough additive or ingredient comprises a vegetable oil, avegetable fat, an animal fat, shortening, butterfat, glycerol or milkfat.
 15. A dough improving composition according to claim 10 wherein thedough improving composition comprises at least one further doughadditive or ingredient and wherein the further dough additive oringredient is a hard wheat flour.
 16. A method of preparing a breadproduct comprising adding a dough improving composition according toclaim 10 to dough ingredients, dough additives or a dough and baking thedough comprising the dough improving composition to obtain the breadproduct.
 17. A dough improving composition according to claim 10 whereinat least one further enzyme is added to the dough improving composition.18. A dough improving composition according to claim 9 wherein at leastone further enzyme is added to the dough improving composition andwherein the further enzyme comprises a xylanase, a cellulase, ahemicellulase, a starch degrading enzyme, a protease, a lipoxygenase, anoxidoreductase or a lipase.
 19. A method of improving the rheologicaland/or machineability properties of a flour dough comprising adding tothe dough a dough improving composition of claim
 10. 20. A method ofimproving the volume or a baked product made from a flour doughcomprising adding to the dough a dough improving composition of claim10.
 21. A method of improving the rheological and/or machineabilityproperties of a flour dough and/or the quality of the product made fromthe dough, comprising adding to the dough a combination comprising ahexose oxidase and a triacylglycerol lipase.
 22. A method of improvingthe rheological and/or machineability properties of a flour dough and/orthe quality of the product made from the dough, comprising adding to thedough a combination comprising a hexose oxidase and a galactolipase. 23.A method of improving the rheological and/or machineability propertiesof a flour dough and/or the quality of the product made from the dough,comprising adding to the dough a combination comprising a hexose oxidaseand a phospholipase.
 24. A method according to claim 1 wherein at leastone further enzyme is added to the dough and wherein the further enzymecomprises a xylanase, an amylase or a mixture of a xylanase and anamylase.
 25. A dough improving composition according to claim 1 whereinthe hexose oxidase is isolated from red algae and wherein the red algaecomprises Iridophycus flaccidum, Chondrus crispus, or Euthora cristata.26. A dough improving composition of claim 10 wherein at least onefurther enzyme is added to the dough improving composition and whereinthe further enzyme comprises a xylanase, an amylase or a mixture of axylanase and an amylase.
 27. A method according to claim 1, wherein thehexose oxidase is isolated from a red algae and wherein the red algaecomprises Iridophycus flaccidum, Chondrus crispus or Euthora cristata.28. A method of improving the rheological and/or machineabilityproperties of a flour dough and/or the quality of the product made fromthe dough, comprising adding to the dough a combination comprising ahexose oxidase and an emulsifying agent; wherein said flour doughcomprises flour, water and at least one further dough additive oringredient; wherein said further dough additive or ingredient isselected from the group consisting of a vegetable oil, a vegetable fat,an animal fat, shortening, butterfat, glycerol, milk fat and a mixturethereof and wherein said further dough additive or ingredient is presentin an amount from 1 to 5% by the weight of the flour component of thedough.
 29. A method according to claim 28 wherein the emulsifying agentis a lipase.
 30. A method according to claim 28 wherein the emulsifyingagent is a lipase and wherein the lipase comprises a triacylglycerollipase, a galactolipase, or a phospholipase.
 31. A method according toclaim 28 wherein the hexose oxidase is isolated from red algae.
 32. Amethod according to claim 28 wherein the flour dough comprises at leastone further dough additive or ingredient.
 33. A method according toclaim 28 wherein the flour dough comprises at least one further doughadditive or ingredient and wherein the further dough additive oringredient is a hard flour.
 34. A method according to claim 28 whereinthe product is a bread product.
 35. A method according to claim 28wherein at least one further enzyme is added to the dough.
 36. A methodaccording to claim 28 wherein at least one further enzyme is added tothe dough and wherein the further enzyme is selected from the groupconsisting of a xylanase, a cellulase, a hemicellulase, a starchdegrading enzyme, a protease, a lipoxygenase, an oxidoreductase, alipase and a mixture thereof.
 37. A dough comprising a dough improvingcomposition wherein said dough improving composition comprises a hexoseoxidase, an emulsifying agent and a further dough additive oringredient; wherein said dough comprises flour and water; wherein saidfurther dough additive or ingredient is selected from the groupconsisting of a vegetable oil, a vegetable fat, an animal fat,shortening, butterfat, glycerol, milk fat and a mixture thereof andwherein said further dough additive or ingredient is present in anamount of from 1 to 5% by weight of the flour component of the dough.38. A dough according to claim 37 wherein the emulsifying agent is alipase.
 39. A dough according to claim 37 wherein the emulsifying agentis a lipase and wherein the lipase comprises a triacylglycerol lipase, agalactolipase, or a phospholipase.
 40. A dough according to claim 37wherein the hexose oxidase is isolated from red algae.
 41. A doughaccording to claim 37 wherein the dough improving composition comprisesat least one further dough additive or ingredient.
 42. A dough accordingto claim 37 wherein the dough improving composition comprises at leastone further dough additive or ingredient and wherein the further doughadditive or ingredient is a hard wheat flour.
 43. A dough according toclaim 37 wherein at least one further enzyme is added to the doughingredients, dough additives or the dough.
 44. A dough according toclaim 37 wherein at least one further enzyme is added to the doughingredients, dough additives or the dough and wherein the further enzymeis selected from the group consisting of a xylanase, an amylase, acellulase, a hemicellulase, a starch degrading enzyme, a protease, alipoxygenase, an oxidoreductase, a lipase, and a mixture thereof.
 45. Adough according to claim 37 wherein at least one further enzyme is addedto the dough ingredients, dough additives or the dough and wherein thefurther enzyme includes a xylanase, an amylase or a mixture thereof. 46.A method for improving a flour dough or the quality of a product madefrom dough, said method comprising adding to the dough a combinationcomprising a hexose oxidase isolated from a red algae and an emulsifyingagent.
 47. The method of claim 46 wherein the red algae is Iridophycusflaccidum.
 48. The method of claim 46 wherein the red algae is Chondruscrispus.
 49. The method of claim 46 wherein the red algae is Euthoracristata.
 50. A dough improving composition comprising a hexose oxidaseisolated from a red algae and an emulsifying agent.
 51. The doughimproving composition of claim 50 wherein the red algae is Iridophycusflaccidum.
 52. The dough improving composition of claim 50 wherein thered algae is Chondrus crispus.
 53. The dough improving composition ofclaim 50 wherein the red algae is Euthora cristata.
 54. A method forimproving a flour dough or the quality of a product made from dough,said method comprising adding to the dough a combination comprising ahexose oxidase and an emulsifying agent comprising an enzyme.
 55. Themethod of claim 54 wherein the enzyme is a lipase.
 56. A dough improvingcomposition comprising a hexose oxidase and an emulsifying agentcomprising an enzyme.